Melanoma differentiation associated gene - 5 and promoter and uses thereof

ABSTRACT

The invention provides for an isolated nucleic acid encoding Mda-5 (melanoma differentiation associated gene-5) and an isolated Mda-5 polypeptide. The invention further provides a vector comprising the nucleic acid encoding Mda-5, as well as a host cell comprising the vector. The invention provides an antibody which specifically binds to an Mda-5 polypeptide. The invention further provides a method for determining whether a compound is an inducer of Mda-5 gene expression and assays to determine whether a compound modifies the enzymatic activity of the Mda-5 polypeptide.

This application is a continuation of U.S. Ser. No. 09/515,363, filedFeb. 29, 2000, the contents of which are hereby incorporated byreference.

The invention disclosed herein was made with Government support underNational Institutes of Health Chemow Endowment No. CA 74468-01 from theU.S. Department of Health and Human Services. Accordingly, the U.S.Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

Throughout this application, various publications are referenced byauthor and date within the text. Full citations for these publicationsmay be found listed alphabetically at the end of the specificationimmediately preceding the claims. All patents, patent applications andpublications cited herein, whether supra or infra, are herebyincorporated by reference in their entirety. The disclosures of thesepublications in their entireties are hereby incorporated by referenceinto this application in order to more fully describe the state of theart as known to those skilled therein as of the date of the inventiondescribed and claimed herein.

Abnormalities in differentiation are common occurrences in human cancers((1) Fisher and Grant, 1985; (2) Waxman, 1995). Moreover, as cancercells evolve, ultimately developing new phenotypes or acquiring afurther elaboration of preexisting transformation-related properties,the degree of expression of differentiation-associated traits oftenundergo a further decline. These observations have been exploited as anovel means of cancer therapy in which tumor cells are treated withagents that induce differentiation and a loss of cancerous properties, astrategy called ‘differentiation therapy’ ((2-4) Waxman et al., 1988,1991; Jiang et al., 1994; Waxman, 1995). In principle, differentiationtherapy may prove less toxic than currently employed chemotherapeuticapproaches, including radiation and treatment with toxic chemicals. Theability to develop rational schemes for applying differentiation therapyclinically require appropriate in vitro and in vivo model systems foridentifying and characterizing the appropriate agent or agents that canmodulate differentiation in cancer cells without causing undue toxicityto normal cells.

SUMMARY OF THE INVENTION

The invention provides for an isolated nucleic acid encoding Mda-5polypeptide as shown in SEQ ID NO:1. A polypeptide having the sequenceshown in SEQ ID NO:2.

The present invention provides for an isolated Mda-5 promoter capable ofdirecting transcription of a heterologous coding sequence positioneddownstream therefrom, wherein the promoter is selected from the groupconsisting of: (a) a promoter comprising the nucleotide sequence shownin SEQ ID NO:3; (b) a promoter comprising a nucleotide sequencefunctionally equivalent to the nucleotide sequence shown in SEQ ID NO:3; and (c) a promoter comprising a nucleotide sequence that hybridizesto a sequence complementary to the promoter of (a) or (b) in a Southernhybridization reaction performed under stringent conditions. Theinvention provides for a host cell comprising the recombinant expressionconstruct as described herein. The invention provides for a method forexpressing foreign DNA in a host cell comprising: introducing into thehost cell a gene transfer vector comprising an Mda-5 promoter nucleotidesequence operably linked to a foreign DNA encoding a desired polypeptideor RNA, wherein said foreign DNA is expressed. The invention furtherprovides for a method for treating cancer in a subject sufferingtherefrom which comprises administering to the subject an effectiveamount of a pharmaceutical composition which comprises a recombinantexpression construct comprising: (a) a nucleic acid molecule thatencodes a selected polypeptide; and (b) an Mda-5 promoter nucleotidesequence operably linked to the nucleic acid molecule of element (a),wherein the coding sequence will be transcribed and translated when in ahost cell to produce the selected polypeptide, and the Mda-5 promoter isheterologous to the coding sequence and a pharmaceutically acceptablecarrier.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1D. Sequence of mda-5 and alignment with CARD and RNAhelicases. FIG. 1A. Nucleotide sequence (SEQ ID NO:1) and correspondingamino acid sequence (SEQ ID NO:2) of mda-5. Underlined sequences areAUUUA sequences. Bold face sequence is the poly A signal. FIG. 1B.Additional nucleotide sequence of mda-5p (SEQ ID NO: 4). Poly A signalis bold faced. FIG. 1C. Alignment of CARD proteins with 50 amino acidsnear the N-terminal region of MDA-5 (a.a. 125-174 correspond to 1-50).(SEQ ID NOS: 5-11) FIG. 1D. Alignment of the RNA helicase conservedmotif of mda-5 with eIF-4A (SEQ ID NO: 12) and p68 RNA helicases-2E (SEQID NO: 13).

FIGS. 2A-2B. Northern blot analysis of mda-5 expression by variouscompounds inducing differentiation in HO-1 human melanoma cells. RNAsamples were extracted from cells treated as indicated for 24 hr. FIG.2A. HO-1 human melanoma cells. FIG. 2B. Early passage human skinfibroblast cells. Northern hybridization was performed as in Materialsand Methods. Abbreviations and concentration of the indicated reagentsare as follows: ctl, control; DMSO, 0.1% dimethyl sulfoxide; EtOH, 0.25%final concentration of ethanol; Mez, mezerein 10 ng/ml; IFN-β, 2,000U/ml interferon-D; IFN-β+Mez, 2,000 U/ml interferon-0 plus mezerein 10ng/ml; IFN-γ, interferon-γ 100 U/ml; IFN-γ+Mez, interferon-γ 100 U/mlplus mezerein 10 ng/ml; RA, all-trans-retinoic acid 2.5 B5M (dissolvedin EtOH): MPA, mycophenolic acid 3 B5M;TPA,12-O-tetradecanoylphorbol-13-acetate 16 nM; cAMP, 3′-5′ cyclicadenosine monophosphate 1 mM; 8-Br-cAMP, 8-bromo-3′-5′ cyclic adenosinemonophosphate 1 mM; 8-Br-cAMP, 8-bromo-3′-5′ cyclic adenosinemonophosphate 1 mM; MMS, methylmethane sulfonate 10 ng/ml; poly IC 10μg/ml.

FIG. 3. Northern blot analysis of mda-5 expression induced by IFN-β innormal and tumor cell lines. RNA samples were extracted from theindicated cells treated with 2,000 U/ml of interferon-β for 24 hr.Northern hybridization was performed as in Materials and Methods.

FIGS. 4A-4B. Northern blot analysis of mda-5 expression by ligands forvarious membrane receptors. RNA samples were extracted from cellstreated as indicated for 24 hr. FIG. 4A. HO-1 human melanoma cells. FIG.4B. Early passage human skin fibroblast cells. Northern hybridizationwas performed as in Materials and Methods. Abbreviations andconcentrations of indicated reagents are as follows: ctl, control;IFN-α, 1,000 U/ml interferon-α IFN-β, 1,000 U/ml interferon-β IFN-γ,1,000 U/ml interferon-γ, IL-6, interleukin-6, 1 ng/ml; EGF, epidermalgrowth factor, 10 ng/ml; TGF-α, transforming growth factor α, 10 ng/ml;TGF-β transforming growth factor β, 2.5 ng/ml; TNF-α, tumor necrosisfactor α, 10 ng/ml; PDGF, platelet-derived growth factor, 10 ng/ml.

FIG. 5. Northern blot analysis and time course of mda-5 expression. RNAsamples were extracted from HO-1 cells treated with the indicatedreagents and harvested at the indicated time after treatment. Northernblotting was performed as in Materials and Methods. Abbreviations andconcentrations of the indicated reagents are as follows: Mez, mezerein10 ng/ml; IFN-β, 2,000 U/ml interferon-β; IFN-5+ Mez, 2,000 U/mlinterferon-β plus mezerein 10 ng/ml.

FIG. 6. Northern blot analysis of mda-5 expression in different organs.Multiple tissue Northern blots were purchased from ClonTech. Each lanecontains 2 μg of poly A+ RNA. Northern hybridization was performed asdescribed in Materials and Methods.

FIGS. 7A-7C. Mechanism of induction of mda-5 expression. A. Northernblot analysis of mda-5. HO-1 melanoma cells were treated with 5 μg/mlactinomycin D 24 hr after incubation with 2,000 U/ml IFN-β or 2,000 U/mlIFN-β+10 ng/ml Mez. Cells were harvested at the indicated time afteractinomycin D treatment. Northern hybridization was performed as inMaterials and Methods. FIG. 7B. Nuclear run-on assays for determiningmda-5 transcription rates. Nuclei were prepared from HO-1 melanoma cellstreated with the indicated reagent(s) for 4 hr. Blots were prepared andhybridized as described in Materials and Methods. Abbreviations andconcentrations of the indicated reagents are as follows: mda-5 5′ and 3′fragment of mda-5 cDNA, respectively; ctl, control; Mez, mezerein 10ng/ml; IFN-β, 2,000 U/ml interferon-β; IFN-β+Mez, 2,000 U/mlinterferon-β plus mezerein 10 ng/ml. FIG. 7C. Northern blot analysis ofmda-5 expression after blocking protein synthesis by cycloheximide(CHX). RNA samples were extracted from HO-1 melanoma cells pretreatedwith 50 μg/ml cycloheximide for 30 min and treated with the indicatedreagents for 8 hr. Abbreviations and concentrations of indicatedreagents are as in FIG. 4.

FIGS. 8A-8C. Protein expression of mda-5. FIG. 8A. Autoradiogram of 9%SDS-PAGE of in vitro translated mda-5 cDNA. β-galactosidase was used asa positive control. FIG. 8B. Western blot analysis of mda-5 fusionprotein resolved in 9% SDS-PAGE. Protein extracts were prepared from293T cells transiently transfected with the indicated expression vector.Details of transfection and immunoblot can be found in Materials andMethods. FIG. 8C. Intracellular localization of mda-5 protein.Transiently transfected 293T cells with the indicated fusion proteinconstructs were mounted and observed by fluorescent confocal microscopy(400×).

FIG. 9. The effect of ectopic expression of mda-5 on G418-resistantcolony formation of HO-1 melanoma cells. HO-1 melanoma cells weretransfected and selected with G418 as in Materials and Methods.Giemsa-stained colonies containing more than about 50 cells werecounted. The results are mean±standard error from three independenttrnasfections (three plates for each transfection) with two differentplasmid batches.

FIG. 10: The sequence of the proximal promoter region of the mda-5 geneshowing landmark restriction sites. The initiator Methionine codon ishighlighted by an open box as is the BstXI sites used to perform aninternal deletion that removed the ATG as described in the text.

FIG. 11: Screening of stable human HO-1 melanoma clones for promoteractivity of stably integrated mda-5 reporter construct. Transfected HO-1cells were selected by Puromycin drug selection and individual coloniesanalyzed for induction of luciferase activity in the presence of IFN-β.Values are expressed as fold change against uninduced values ofluciferase activity.

FIG. 12: Induction kinetics of mda-5 promoter activity. Stable clones#20 and #40 were treated with IFN-β and samples were harvested andanalyzed for luciferase activity at the times indicated.

FIG. 13: Responsiveness of the mda-5 promoter to IFN-β levels: Stableclones #20 and #40 were treated with IFN-β and samples were harvestedand analyzed for luciferase activity 48 h after initiation of treatment.The extent of activity was normalized based on equivalent proteincontent and performed in duplicate for each clone.

FIGS. 14A-14B: Responsiveness of the mda-5 promoter to various inducers:FIG. 14A. HO-1 cells transiently transfected with the mda-5 reporter andtreated for 48 h with equivalent units of IFNs α, β and γ and TNF-α andpoly IC:IC. The luciferase activity was expressed as fold increase overuntreated control cells. FIG. 14B. Clone #40 was treated with equivalentunits of the indicated IFNs for 48 h and luciferase activity expressedas fold activation over untreated cells determined.

FIG. 15: Induction kinetics of mda-5 promoter activity by doublestranded RNA. Stable clones #20 and #40 were treated with 2 μg/ml polyIC:IC and samples harvested and analyzed for luciferase activity at thetimes indicated.

DETAILED DESCRIPTION OF THE INVENTION

The following abbreviations are used herein: Mda-5—Melanomadifferentiation associated gene-5, CMV—cytomegalovirus,

The invention provides for an isolated nucleic acid comprising thesequence shown in SEQ ID NO: 1 encoding a Melanoma DifferentiationAssociated Gene-5 (Mda-5) polypeptide.

In one embodiment, the invention provides for an isolated nucleic acidcomprising a derivative of the sequence of SEQ ID NO:1 encoding apolypeptide which is functionally equivalent to Mda-5.

The present invention also provides for a fragment of the isolatednucleic acid aforementioned, wherein the fragment encodes a polypeptidehaving Mda-5 biological activity.

The invention provides for a nucleic acid which hybridizes to the DNAshown in SEQ ID NO:1 or the complementary strand thereof, wherein thenucleic acid or the complementary strand thereof, encodes a polypeptidehaving Mda-5 activity.

The invention further provides for a vector comprising any of thenucleic acids described herein. In one embodiment, the vector is areplicable vector, a gene transfer vector, an expression vector, or avector capable of driving expression of a gene of interest in a hostcell.

The invention provides for a host cell comprising the aforementionedvector.

The invention provides a method for identifying a compound as an agonistor antagonist of interferon-β, interferon-α or interferon γ whichcomprises: (a) contacting a cell with the compound, wherein the cellcomprises a nucleic acid having the sequence shown in SEQ ID NO:2, or afunctional equivalent thereof, operably linked to a reporter gene; (b)measuring the level of reporter gene expressed by the cell in thepresence of the compound; (c) comparing the expression level of thereporter gene measured in step (b) with the expression level of reportergene measured in the absence of the compound, so as to identify whetherthe compound is an interferon agonist or antagonist; wherein a higherlevel of reporter gene expression measured in step (b) is indicative ofthe compound being an interferon agonist, and wherein a lower level ofreporter gene expression measured in step (b) is indicative of thecompound being an interferon antagonist.

In one embodiment, the compound is a small organic molecule having aweight of about 5 kilodaltons or less.

In another embodiment, the cell is a HO-1 human melanoma cell.

In another embodiment of the invention, the level of reporter geneexpression measured which is indicative of an agonist is from 10 to 1000fold higher than the level of reporter gene expression measured in theabsence of the compound.

In another embodiment of the invention, the reporter gene is luciferase.

The invention provides for an isolated polypeptide having the amino acidsequence shown in SEQ ID NO:2 encoding Mda-5.

The invention also provides for an isolated antibody which specificallybinds to the polypeptide having the sequence shown in SEQ ID NO:2.

In one embodiment, the antibody is a monoclonal antibody.

The invention provides for an isolated Mda-5 promoter capable ofdirecting transcription of a heterologous coding sequence positioneddownstream therefrom, wherein the promoter is selected from the groupconsisting of: (a) a promoter comprising the nucleotide sequence shownin SEQ ID NO:3; (b) a promoter comprising a nucleotide sequencefunctionally equivalent to the nucleotide sequence shown in SEQ ID NO:3; and (c) a promoter comprising a nucleotide sequence that hybridizesto a sequence complementary to the promoter of (a) or (b) in a Southernhybridization reaction performed under stringent conditions.

In one embodiment, the promoter comprises the nucleotide sequence shownin SEQ ID NO:3.

The invention provides for a recombinant expression construct effectivein directing the transcription of a selected coding sequence whichcomprises: (a) an Mda-5 promoter nucleotide sequence according to claim15; and (b) a coding sequence operably linked to the promoter, wherebythe coding sequence can be transcribed and translated in a host cell,and the promoter is heterologous to the coding sequence.

In one embodiment, the Mda-5 promoter comprises a human Mda-5 promoter.

In another embodiment, the human Mda-5 promoter comprises the nucleotidesequence shown in SEQ ID NO:3.

In another embodiment, the coding sequence encodes a tumor suppressorpolypeptide.

In another embodiment, the tumor suppressor polypeptide is p21,retinoblastoma protein or p53.

The invention provides for a host cell comprising the recombinantexpression construct described herein. In one embodiment the host cellis stably transformed with the recombinant expression construct.

In another embodiment, the host cell is a tumor cell.

In another embodiment, the host cell is a melanocyte.

In another embodiment, the cell is an immortalized cell.

In another embodiment, the tumor cell is a melanoma cell, aneuroblastoma cell, an astrocytoma cell, a glioblastomoa multifore cell,a cerival cancer cell, a breast cancer cell, a lung cancer cell or aprostate cancer cell.

The invention provides for an isolated Mda-5 promoter capable ofdirecting the transcription of a heterologous coding sequence positioneddownstream therefrom, wherein the promoter is selected from the groupconsisting of (a) a promoter comprising the nucleotide sequence shown inSEQ ID NO:3; (b) a promoter comprising a nucleotide sequencefunctionally equivalent to the promoter in element (a); and (c) apromoter comprising a nucleotide sequence that hybridizes to a sequencecomplementary to the promoter of element (a) or element (b) in aSouthern hybridization reaction performed under stringent conditions.

The invention also provides for a method for treating cancer in asubject suffering therefrom which comprises administering to the subjectan effective amount of a pharmaceutical composition which comprises arecombinant expression construct comprising:

-   -   (a) a nucleic acid molecule that encodes a polypeptide of        interest; and    -   (b) an Mda-5 promoter nucleotide sequence operably linked to the        nucleic acid molecule of element (a), and wherein the Mda-5        promoter is heterologous to the nucleic acid molecule,    -   and a pharmaceutically acceptable carrier.

In one embodiment, the cancer is melanoma, neuroblastoma, astrocytoma,glioblastoma multiforme, cervical cancer, breast cancer, colon cancer,prostate cancer, osteoscarcoma, or chrondosarcoma.

In one embodiment, the cancer is a cancer of the central nervous systemof the subject.

In one embodiment, the administering is carried out via injection, oraladministration, topical administration, adenovirus infection,liposome-mediated transfer, topical application to the cells of thesubject, or microinjection.

In one embodiment, the carrier is an aqueous carrier, a liposome, or alipid carrier. mda-5 cDNA (SEQ ID NO:1) GCGCGCCGGC CTGAGAGCCC TGTGGACAACCTCGTCATTG TCAGGCACAG AGCGGTAGAC CCTGCTTCTC TAAGTGGGCA GCGGACAGCGGCACGCACAT TTCACCTGTC CCGCAGACAA CAGCACCATC TGCTTGGGAG AACCCTCTCCCTTCTCTGAG AAAGAAAGAT GTCGAATGGG TATTCCACAG ACGAGAATTT CCGCTATCTCATCTCGTGCT TCAGGGCCAG GGTGAAAATG TACATCCAGG TGGAGCCTGT GCTGGACTACCTGACCTTTC TGCCTGCAGA GGTGAAGGAG CAGATTCAGA GGACAGTCGC CACCTCCGGGAACATGCAGG CAGTTGAACT GCTGCTGAGC ACCTTGGAGA AGGGAGTCTG GCACCTTGGTTGGACTCGGG AATTCGTGGA GGCCCTCCGG AGAACCGGCA GCCCTCTGGC CGCCCGCTACATGAACCCTG AGCTCACGGA CTTGCCCTCT CCATCGTTTG AGAACGCTCA TGATGAATATCTCCAACTGC TGAACCTCCT TCAGCCCACT CTGGTGGACA AGCTTCTAGT TAGAGACGTCTTGGATAAGT GCATGGAGGA GGAACTGTTG ACAATTGAAG ACAGAAACCG GATTGCTGCTGCAGAAAACA ATGGAAATGA ATCAGGTGTA AGAGAGCTAC TAAAAAGGAT TGTGCAGAAAGAAAACTGGT TCTCTGCATT TCTGAATGTT CTTCGTCAAA CAGGAAACAA TGAACTTGTCCAAGAGTTAA CAGGCTCTGA TTGCTCAGAA AGCAATGCAG AGATTGAGAA TTTATCACAAGTTGATGGTC CTCAAGTGGA AGAGCAACTT CTTTCAACCA CAGTTCAGCC AAATCTGGAGAAGGAGGTCT GGGGCATGGA GAATAACTCA TCAGAATCAT CTTTTGCAGA TTCTTCTGTAGTTTCAGAAT CAGACACAAG TTTGGCAGAA GGAAGTGTCA GCTGCTTAGA TGAAAGTCTTGGACATAACA GCAACATGGG CAGTGATTCA GGCACCATGG GAAGTGATTC AGATGAAGAGAATGTGGCAG CAAGAGCATC CCCGGAGCCA GAACTCCAGC TCAGGCCTTA CCAAATGGAAGTTGCCCAGC CAGCCTTGGA AGGGAAGAAT ATCATCATCT GCCTCCCTAC AGGGAGTGGAAAAACCAGAG TGGCTGTTTA CATTGCCAAG GATCACTTAG ACAAGAAGAA AAAAGCATCTGAGCCTGGAA AAGTTATAGT TCTTGTCAAT AAGGTACTGC TAGTTGAACA GCTCTTCCGCAAGGAGTTCC AACCATTTTT GAAGAAATGG TATCGTGTTA TTGGATTAAG TGGTGATACCCAACTGAAAA TATCATTTCC AGAAGTTGTC AAGTCCTGTG ATATTATTAT CAGTACAGCTCAAATCCTTG AAAACTCCCT CTTAAACTTG GAAAATGGAG AAGATGCTGG TGTTCAATTGTCAGACTTTT CCCTCATTAT CATTGATGAA TGTCATCACA CCAACAAAGA AGCAGTGTATAATAACATCA TGAGGCATTA TTTGATGCAG AAGTTGAAAA ACAATAGACT CAAGAAAGAAAACAAACCAG TGATTCCCCT TCCTCAGATA CTGGGACTAA CAGCTTCACC TGGTGTTGGAGGGGCCACGA AGCAAGCCAA AGCTGAAGAA CACATTTTAA AACTATGTGC CAATCTTGATGCATTTACTA TTAAAACTGT TAAAGAAAAC CTTGATCAAC TGAAAAACCA AATACAGGAGCCATGCAAGA AGTTTGCCAT TGCAGATGCA ACCAGAGAAG ATCCATTTAA AGAGAAACTTCTAGAAATAA TGACAAGGAT TCAAACTTAT TGTCAAATGA GTCCAATGTC AGATTTTGGAACTCAACCCT ATGAACAATG GGCCATTCAA ATGGAAAAAA AAGCTGCAAA AAAAGGAAATCGCAAAGAAC GTGTTTGTGC AGAACATTTG AGGAAGTACA ATGAGGCCCT ACAAATTAATGACACAATTC GAATGATAGA TGCGTATACT CATCTTGAAA CTTTCTATAA TGAAGAGAAAGATAAGAAGT TTGCAGTCAT AGAAGATGAT AGTGATGAGG GTGGTGATGA TGAGTATTGTGATGGTGATG AAGATGAGGA TGATTTAAAG AAACCTTTGA AACTGGATGA AACAGATAGATTTCTCATGA CTTTATTTTT TGAAAACAAT AAAATGTTGA AAAGGCTGGC TGAAAACCCAGAATATGAAA ATGAAAAGCT GACCAAATTA AGAAATACCA TAATGGAGCA ATATACTAGGACTGAGGAAT CAGCACGAGG AATAATCTTT ACAAAAACAC GACAGAGTGC ATATGCGCTTTCCCAGTGGA TTACTGAAAA TGAAAAATTT GCTGAAGTAG GAGTCAAAGC CCACCATCTGATTGGAGCTG GACACAGCAG TGAGTTCAAA CCCATGACAC AGAATGAACA AAAAGAAGTCATTAGTAAAT TTCGCACTGG AAAAATCAAT CTGCTTATCG CTACCACAGT GGCAGAAGAAGGTCTGGATA TTAAAGAATG TAACATTGTT ATCCGTTATG GTCTCGTCAC CAATGAAATAGCCATGGTCC AGGCCCGTGG TCGAGCCAGA GCTGATGAGA GCACCTACGT CCTGGTTGCTCACAGTGGTT CAGGAGTTAT CGAACATGAG ACAGTTAATG ATTTCCGAGA GAAGATGATGTATAAAGCTA TACATTGTGT TCAAAATATG AAACCAGAGG AGTATGCTCA TAAGATTTTGGAATTACAGA TGCAAAGTAT AATGGAAAAG AAAATGAAAA CCAAGAGAAA TATTGCCAAGCATTACAAGA ATAACCCATC ACTAATAACT TTCCTTTGCA AAAACTGCAG TGTGCTAGCCTGTTCTGGGG AAGATATCCA TGTAATTGAG AAAATGCATC ACGTCAATAT GACCCCAGAATTCAAGGAAC TTTACATTGT AAGAGAAAAC AAAGCACTGC AAAAGAAGTG TGCCGACTATCAAATAAATG GTGAAATCAT CTGCAAATGT GGCCAGGCTT GGGGAACAAT GATGGTGCACAAAGGCTTAG ATTTGCCTTG TCTCAAAATA AGGAATTTTG TAGTGGTTTT CAAAAATAATTCAACAAAGA AACAATACAA AAAGTGGGTA GAATTACCTA TCACATTTCC CAATCTTGACTATTCAGAAT GCTGTTTATT TAGTGATGAG GATTAGCACT TGATTGAAGA TTCTTTTAAAATACTATCAG TTAAACATTT AATATGATTA TGATTAATGT ATTCATTATG CTACAGAACTGACATAAGAA TCAATAAAAT GATTGTTTTA CTCTG Mda-5 potein sequence (SEQ IDNO:2) MSNGYSTDEN FRYLISCFRA RVKMYIQVEP VLDYLTFLPA EVKEQIQRTV ATSGNMQAVELLLSTLEKGV WHLGWTREFV EALRRTGSPL AARYMNPELT DLPSPSFENA HDEYLQLLNLLQPTLVDKLL VRDVLDKCME EELLTIEDRN RIAAAENNGN ESGVRELLKR IVQKENWFSAFLNVLRQTGN NELVQELTGS DCSESNAEIE NLSQVDGPQV EEQLLSTTVQ PNLEKEVWGMENNSSESSFA DSSVVSESDT SLAEGSVSCL DESLGHNSNN GSDSGTMGSD SDEENVAARASPEPELQLRP YQMEVAQPAL EGKNIIICLP TGSGKTRVAV YIAKDHLDKK KKASEPGKVIVLVNKVLLVE QLFRKEFQPF LKKWYRVIGL SGDTQLKISF PEVVKSCDII ISTAQILENSLLNLENGEDA GVQLSDFSLI IIDECHHTNK EAVYNNIMRH YLMQKLKNNR LKKENKPVIPLPQILGLTAS PGVGGATKQA KAEEHILKLC ANLDAFTIKT VKENLDQLKN QIQEPCKKFAIADATREDPF KEKLLEIMTR IQTYCQMSPM SDFGTQPYEQ WAIQMEKKAA KKGNRKERVCAEHLRKYNEA LQINDTIRMI DAYTHLETFY NEEKDKKFAV IEDDSDEGGD DEYCDGDEDEDDLKKPLKLD ETDRFLMTLF FENNKMLKRL AENPEYENEK LTKLRNTIME QYTRTEESARGIIFTKTRQS AYALSQWITE NEKFAEVGVK AHHLIGAGHS SEFKPMTQNE QKEVISKFRTGKINLLIATT VAEEGLDIKE CNIVIRYGLV TNEIAMVQAR GRARADESTY VLVAHSGSGVIEHETVNDFR EKMMYKAIHC VQNMKPEEYA HKILELQMQS IMEKKMKTKR NIAKHYKNNPSLITFLCKNC SVLACSGEDI HVIEKMHHVN MTPEFKELYI VRENKALQKK CADYQINGEIICKCGQAWGT MMVHKGLDLP CLKIRNFVVV FKNNSTKKQY KKWVELPITF PNLDYSECCL FSDED•

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiology,virology, recombinant DNA technology, and immunology, which are withinthe skill of the art. Such techniques are explained fully in theliterature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning:A Laboratory Manual, Second Edition (1989); DNA Cloning, Vols. I and II(D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed.1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds.1984); Animal Cell Culture (R. K. Freshney ed. 1986); Immobilized Cellsand Enzymes (IRL press, 1986); Perbal, B., A Practical Guide toMolecular Cloning (1984); the series, Methods In Enzymology (S. Colowickand N. Kaplan eds., Academic Press, Inc.); and Handbook of ExperimentalImmunology, Vols. I-IV (D. M. Weir and C. C. Blackwell eds., 1986,Blackwell Scientific Publications).

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural references unless the contentclearly dictates otherwise.

The present invention provides for an isolated Mda-5 promoter capable ofdirecting transcription of a heterologous coding sequence positioneddownstream therefrom, wherein the promoter is selected from the groupconsisting of: (a) a promoter comprising the nucleotide sequence shownin SEQ ID NO: 3; (b) a promoter comprising a nucleotide sequencefunctionally equivalent to the nucleotide sequence shown in SEQ ID NO:3; and (c) a promoter comprising a nucleotide sequence that hybridizesto a sequence complementary to the promoter of (a) or (b) in a Southernhybridization reaction performed under stringent conditions.

In one embodiment of the invention, the promoter comprises thenucleotide sequence shown in SEQ ID NO: 3.

The present invention also provides for a recombinant expressionconstruct effective in directing the transcription of a selected codingsequence which comprises:

(a) an Mda-5 promoter nucleotide sequence as described herein; and

(b) a coding sequence operably linked to the promoter, whereby thecoding sequence can be transcribed and translated in a host cell, andthe promoter is heterologous to the coding sequence. In anotherembodiment of the invention, the Mda-5 promoter comprises a human Mda-5promoter.

In another embodiment of the invention, the human Mda-5 promotercomprises the nucleotide sequence shown in SEQ ID NO:3.

In another embodiment of the invention, the coding sequence encodes atumor suppressor polypeptide.

In another embodiment of the invention, the tumor suppressor polypeptideis p21, retinoblastoma protein or p53.

The invention provides for a host cell comprising the recombinantexpression construct as described herein.

In another embodiment of the invention, the host cell is stablytransformed with the recombinant expression construct described herein.

In another embodiment of the invention, the host cell is a tumor cell.

In another embodiment of the invention, the host cell is a melanocyte.

In another embodiment of the invention, the cell is an immortalizedcell.

In another embodiment of the invention, the tumor cell is a melanomacell, a neuroblastoma cell, an astrocytoma cell, a glioblastomoamultifore cell, a cerival cancer cell, a breast cancer cell, a lungcancer cell or a prostate cancer cell.

The invention provides for a method for expressing foreign DNA in a hostcell comprising: introducing into the host cell a gene transfer vectorcomprising an Mda-5 promoter nucleotide sequence operably linked to aforeign DNA encoding a desired polypeptide or RNA, wherein said foreignDNA is expressed.

In another embodiment of the invention, the gene transfer vector encodesand expresses a reporter molecule.

In another embodiment of the invention, the reporter molecule isselected from the group consisting of beta-galactosidase, luciferase andchloramphenicol acetyltransferase.

In another embodiment of the invention, the “introducing” is carried outby a means selected from the group consisting of adenovirus infection,liposome-mediated transfer, topical application to the cell, andmicroinjection.

The invention provides for an isolated Mda-5 promoter capable ofdirecting the transcription of a heterologous coding sequence positioneddownstream therefrom, wherein the promoter is selected from the groupconsisting of (a) a promoter comprising the nucleotide sequence shown inSEQ ID NO:3; (b) a promoter comprising a nucleotide sequencefunctionally equivalent to the promoter in element (a); and (c) apromoter comprising a nucleotide sequence that hybridizes to a sequencecomplementary to the promoter of element (a) or element (b) in aSouthern hybridization reaction performed under stringent conditions.

The invention further provides for a method for treating cancer in asubject suffering therefrom which comprises administering to the subjectan effective amount of a pharmaceutical composition which comprises arecombinant expression construct comprising: (a) a nucleic acid moleculethat encodes a selected polypeptide; and

(b) an Mda-5 promoter nucleotide sequence operably linked to the nucleicacid molecule of element (a), wherein the coding sequence will betranscribed and translated when in a host cell to produce the selectedpolypeptide, and the Mda-5 promoter is heterologous to the codingsequence and a pharmaceutically acceptable carrier.

In another embodiment of the invention, the cancer is melanoma,neuroblastoma, astrocytoma, glioblastoma multiforme, cervical cancer,breast cancer, colon cancer, prostate cancer, osteoscarcoma, orchrondosarcoma.

In another embodiment of the invention, the cancer is a cancer of thecentral nervous system of the subject.

In another embodiment of the invention, the administering is carried outvia injection, oral administration, or topical administration.

In another embodiment of the invention, the carrier is an aqueouscarrier, a liposome, or a lipid carrier.

A method for determining whether a compound is an inducer of Mda-5 geneexpression in a cell and an inducer of terminal differentiation of suchcell which comprises: (a) contacting a cell with the compound, whereinthe cell comprises a nucleic acid encoding Mda-5 having the sequenceshown in SEQ ID NO:1, or a functional equivalent thereof, operablylinked to an Mda-5 promoter; (b) measuring the level of either (i) Mda-5mRNA produced or (ii) Mda-5 polypeptide expressed by the cell in thepresence of the compound; (c) comparing the expression level of Mda-5mRNA or polypeptide measured in step (b) with the level measured in theabsence of the compound, so as to determine whether the compound is aninducer of Mda-5 gene expression and an inducer of terminaldifferentiation of the cell.

A method for treating cancer in a subject suffering therefrom whichcomprises administering to the subject an effective amount of a compoundidentified by the method of identifying an inducer of Mda-5 geneexpression described herein and a pharmaceutically acceptable carrier,so as to induce terminal differentiation of the cancer cells in thesubject and thereby treat the cancer.

Definitions

As used herein “therapeutic gene” means DNA encoding an amino acidsequence corresponding to a functional protein capable of exerting atherapeutic effect on cancer cells or having a regulatory effect on theexpression of a function in cells.

As used herein “nucleic acid molecule” includes both DNA and RNA and,unless otherwise specified, includes both double-stranded andsingle-stranded nucleic acids. Also included are hybrids such as DNA-RNAhybrids. Reference to a nucleic acid sequence can also include modifiedbases as long as the modification does not significantly interfereeither with binding of a ligand such as a protein by the nucleic acid orWatson-Crick base pairing.

As used herein “Mda-5 promoter” means the promoter having about 1000base pairs (bp) derived from the 5′ flanking region of the Mda-5 gene asshown in FIG. 10. See SEQ ID NO:3 as follows.

Mda-5 cDNA (SEQ ID NO:1) and Mda-5 polypeptide (SEQ ID NO:2) mda-5 cDNA(SEQ ID NO:1) GCGCGCCGGC CTGAGAGCCC TGTGGACAAC CTCGTCATTG TCAGGCACAGAGCGGTAGAC CCTGCTTCTC TAAGTGGGCA GCGGACAGCG GCACGCACAT TTCACCTGTCCCGCAGACAA CAGCACCATC TGCTTGGGAG AACCCTCTCC CTTCTCTGAG AAAGAAAGATGTCGAATGGG TATTCCACAG ACGAGAATTT CCGCTATCTC ATCTCGTGCT TCAGGGCCAGGGTGAAAATG TACATCCAGG TGGAGCCTGT GCTGGACTAC CTGACCTTTC TGCCTGCAGAGGTGAAGGAG CAGATTCAGA GGACAGTCGC CACCTCCGGG AACATGCAGG CAGTTGAACTGCTGCTGAGC ACCTTGGAGA AGGGAGTCTG GCACCTTGGT TGGACTCGGG AATTCGTGGAGGCCCTCCGG AGAACCGGCA GCCCTCTGGC CGCCCGCTAC ATGAACCCTG AGCTCACGGACTTGCCCTCT CCATCGTTTG AGAACGCTCA TGATGAATAT CTCCAACTGC TGAACCTCCTTCAGCCCACT CTGGTGGACA AGCTTCTAGT TAGAGACGTC TTGGATAAGT GCATGGAGGAGGAACTGTTG ACAATTGAAG ACAGAAACCG GATTGCTGCT GCAGAAAACA ATGGAAATGAATCAGGTGTA AGAGAGCTAC TAAAAAGGAT TGTGCAGAAA GAAAACTGGT TCTCTGCATTTCTGAATGTT CTTCGTCAAA CAGGAAACAA TGAACTTGTC CAAGAGTTAA CAGGCTCTGATTGCTCAGAA AGCAATGCAG AGATTGAGAA TTTATCACAA GTTGATGGTC CTCAAGTGGAAGAGCAACTT CTTTCAACCA CAGTTCAGCC AAATCTGGAG AAGGAGGTCT GGGGCATGGAGAATAACTCA TCAGAATCAT CTTTTGCAGA TTCTTCTGTA GTTTCAGAAT CAGACACAAGTTTGGCAGAA GGAAGTGTCA GCTGCTTAGA TGAAAGTCTT GGACATAACA GCAACATGGGCAGTGATTCA GGCACCATGG GAAGTGATTC AGATGAAGAG AATGTGGCAG CAAGAGCATCCCCGGAGCCA GAACTCCAGC TCAGGCCTTA CCAAATGGAA GTTGCCCAGC CAGCCTTGGAAGGGAAGAAT ATCATCATCT GCCTCCCTAC AGGGAGTGGA AAAACCAGAG TGGCTGTTTACATTGCCAAG GATCACTTAG ACAAGAAGAA AAAAGCATCT GAGCCTGGAA AAGTTATAGTTCTTGTCAAT AAGGTACTGC TAGTTGAACA GCTCTTCCGC AAGGAGTTCC AACCATTTTTGAAGAAATGG TATCGTGTTA TTGGATTAAG TGGTGATACC CAACTGAAAA TATCATTTCCAGAAGTTGTC AAGTCCTGTG ATATTATTAT CAGTACAGCT CAAATCCTTG AAAACTCCCTCTTAAACTTG GAAAATGGAG AAGATGCTGG TGTTCAATTG TCAGACTTTT CCCTCATTATCATTGATGAA TGTCATCACA CCAACAAAGA AGCAGTGTAT AATAACATCA TGAGGCATTATTTGATGCAG AAGTTGAAAA ACAATAGACT CAAGAAAGAA AACAAACCAG TGATTCCCCTTCCTCAGATA CTGGGACTAA CAGCTTCACC TGGTGTTGGA GGGGCCACGA AGCAAGCCAAAGCTGAAGAA CACATTTTAA AACTATGTGC CAATCTTGAT GCATTTACTA TTAAAACTGTTAAAGAAAAC CTTGATCAAC TGAAAAACCA AATACAGGAG CCATGCAAGA AGTTTGCCATTGCAGATGCA ACCAGAGAAG ATCCATTTAA AGAGAAACTT CTAGAAATAA TGACAAGGATTCAAACTTAT TGTCAAATGA GTCCAATGTC AGATTTTGGA ACTCAACCCT ATGAACAATGGGCCATTCAA ATGGAAAAAA AAGCTGCAAA AAAAGGAAAT CGCAAAGAAC GTGTTTGTGCAGAACATTTG AGGAAGTACA ATGAGGCCCT ACAAATTAAT GACACAATTC GAATGATAGATGCGTATACT CATCTTGAAA CTTTCTATAA TGAAGAGAAA GATAAGAAGT TTGCAGTCATAGAAGATGAT AGTGATGAGG GTGGTGATGA TGAGTATTGT GATGGTGATG AAGATGAGGATGATTTAAAG AAACCTTTGA AACTGGATGA AACAGATAGA TTTCTCATGA CTTTATTTTTTGAAAACAAT AAAATGTTGA AAAGGCTGGC TGAAAACCCA GAATATGAAA ATGAAAAGCTGACCAAATTA AGAAATACCA TAATGGAGCA ATATACTAGG ACTGAGGAAT CAGCACGAGGAATAATCTTT ACAAAAACAC GACAGAGTGC ATATGCGCTT TCCCAGTGGA TTACTGAAAATGAAAAATTT GCTGAAGTAG GAGTCAAAGC CCACCATCTG ATTGGAGCTG GACACAGCAGTGAGTTCAAA CCCATGACAC AGAATGAACA AAAAGAAGTC ATTAGTAAAT TTCGCACTGGAAAAATCAAT CTGCTTATCG CTACCACAGT GGCAGAAGAA GGTCTGGATA TTAAAGAATGTAACATTGTT ATCCGTTATG GTCTCGTCAC CAATGAAATA GCCATGGTCC AGGCCCGTGGTCGAGCCAGA GCTGATGAGA GCACCTACGT CCTGGTTGCT CACAGTGGTT CAGGAGTTATCGAACATGAG ACAGTTAATG ATTTCCGAGA GAAGATGATG TATAAAGCTA TACATTGTGTTCAAAATATG AAACCAGAGG AGTATGCTCA TAAGATTTTG GAATTACAGA TGCAAAGTATAATGGAAAAG AAAATGAAAA CCAAGAGAAA TATTGCCAAG CATTACAAGA ATAACCCATCACTAATAACT TTCCTTTGCA AAAACTGCAG TGTGCTAGCC TGTTCTGGGG AAGATATCCATGTAATTGAG AAAATGCATC ACGTCAATAT GACCCCAGAA TTCAAGGAAC TTTACATTGTAAGAGAAAAC AAAGCACTGC AAAAGAAGTG TGCCGACTAT CAAATAAATG GTGAAATCATCTGCAAATGT GGCCAGGCTT GGGGAACAAT GATGGTGCAC AAAGGCTTAG ATTTGCCTTGTCTCAAAATA AGGAATTTTG TAGTGGTTTT CAAAAATAAT TCAACAAAGA AACAATACAAAAAGTGGGTA GAATTACCTA TCACATTTCC CAATCTTGAC TATTCAGAAT GCTGTTTATTTAGTGATGAG GATTAGCACT TGATTGAAGA TTCTTTTAAA ATACTATCAG TTAAACATTTAATATGATTA TGATTAATGT ATTCATTATG CTACAGAACT GACATAAGAA TCAATAAAATGATTGTTTTA CTCTG Mda-5 potein sequence (SEQ ID NO:2) MSNGYSTDENFRYLISCFRA RVKMYIQVEP VLDYLTFLPA EVKEQIQRTV ATSGNMQAVE LLLSTLEKGVWHLGWTREFV EALRRTGSPL AARYMNPELT DLPSPSFENA HDEYLQLLNL LQPTLVDKLLVRDVLDKCME EELLTIEDRN RIAAAENNGN ESGVRELLKR IVQKENWFSA FLNVLRQTGNNELVQELTGS DCSESNAEIE NLSQVDGPQV EEQLLSTTVQ PNLEKEVWGM ENNSSESSFADSSVVSESDT SLAEGSVSCL DESLGHNSNN GSDSGTMGSD SDEENVAARA SPEPELQLRPYQMEVAQPAL EGKNIIICLP TGSGKTRVAV YIAKDHLDKK KKASEPGKVI VLVNKVLLVEQLFRKEFQPF LKKWYRVIGL SGDTQLKISF PEVVKSCDII ISTAQILENS LLNLENGEDAGVQLSDFSLI IIDECHHTNK EAVYNNIMRH YLMQKLKNNR LKKENKPVIP LPQILGLTASPGVGGATKQA KAEEHILKLC ANLDAFTIKT VKENLDQLKN QIQEPCKKFA IADATREDPFKEKLLEIMTR IQTYCQMSPM SDFGTQPYEQ WAIQMEKKAA KKGNRKERVC AEHLRKYNEALQINDTIRMI DAYTHLETFY NEEKDKKFAV IEDDSDEGGD DEYCDGDEDE DDLKKPLKLDETDRFLMTLF FENNKMLKRL AENPEYENEK LTKLRNTIME QYTRTEESAR GIIFTKTRQSAYALSQWITE NEKFAEVGVK AHHLIGAGHS SEFKPMTQNE QKEVISKFRT GKINLLIATTVAEEGLDIKE CNIVIRYGLV TNEIAMVQAR GRARADESTY VLVAHSGSGV IEHETVNDFREKMMYKAIHC VQNMKPEEYA HKILELQMQS IMEKKMKTKR NIAKHYKNNP SLITFLCKNCSVLACSGEDI HVIEKMHHVN MTPEFKELYI VRENKALQKK CADYQINGEI ICKCGQAWGTMMVHKGLDLP CLKIRNFVVV FKNNSTKKQY KKWVELPITF PNLDYSECCL FSDED• Mda-5promoter sequence (SEQ ID NO:3) GCACATTTTG GCCTACAAAG GACCTTATTGTTAAGGCAGA ACCTGCTGGG AAAACAAAAT ATCCGCCGGA GGAGCTTTGT AGAGCGTTGGTCTTGGTGTC AGAGAGAATT CGCTTTCCTT TTCTGTTTCC CGCGGTGTCC TTAACCAAAGGCCTCCTCTC TTCACCCGCC CCGACCAAAA GGTGGCGTCT CCCTGAGGAA ACTCCCTCCCCGCCAGGCAG ATTACGTTTA CAAAGTCCTG AGAAGAGAAT CGAAACAGAA ACCAAAGTCAGGCAAACTCT GTAAGAACTG CCTGACAGAA AGCTGGACTC AAAGCTCCTA CCCGAGTGTGCAGCAGGATC GCCCCGGTCC GGGACCCCAG GCGCACACCG CAGAGTCCAA AGTGCCGCGCCTGCCGGCCG CACCTGCCTG CCGCGGCCCC GCGCGCCGCC CCGCTGCCCA CCTGCCCGCCTGCCCACCTG CCCAGGTGCG AGTGCAGCCC CGCGCGCCGG CCTGAGAGCC CTGTGGACAACCTCGTCATT GTCAGGCACA GAGCGGTAGA CCCTGCTTCT NTAAGTGGGC AGCGGACAGCGGCACGCACA TTTCACCTGT CCCGCAGACA ACAGCACCAT CTGCTTGGGA GAACCCTCTCCCTTCTCTGA GAAAGAAAGA TGTCGAATGG GTATTCCACA GACGAGAATT TCCGCTATCTCATCTCGTGC TTCAGGGCCA GGGTGAAAAT GTACATCCAG GTGGAGCCTG TGCTGGACTACCTGACCTTT CTGCCTGCAG AGGTGAAGGA GCAGATTCAG AGGACAGTCG CCACCTCCGGGAACATGCAG GCAGTTGAAC TGCTGCTGAG CACCTTGGAG AAGGGAGTCT GGCACCTTGGTTGGACTCGG GAATTCGTGG AGGCCCTCCG GAGAACCGGC AGCCCTCTGG CCGCCCGCTACATGAACCCT GAGCTCACGG ACTTGCCCTC TCCATCGTTT GAGAACGCTC ATGATGAATATCTCCAACTG CTGAACCTCC TTCAGCCCAC TCTGGTGGAC AAGCTT(See also FIG. 10 for the Mda-5 promoter sequence).

As used herein “enhancer element” is a nucleotide sequence thatincreases the rate of transcription of the therapeutic genes or genes ofinterest but does not have promoter activity. An enhancer can be movedupstream, downstream, and to the other side of a promoter withoutsignificant loss of activity.

Two DNA or polypeptide sequences are “substantially homologous” when atleast about 80% (preferably at least about 90%, and most preferably atleast about 95%) of the nucleotides or amino acids match over a definedlength of the molecule. As used herein, “substantially homologous” alsorefers to sequences showing identity to the specified DNA or polypeptidesequence. DNA sequences that are substantially homologous can beidentified in a Southern hybridization, experiment under, for example,stringent conditions, as defined for that particular system. Definingappropriate hybridization conditions is within the skill of the art.See, e.g., Sambrook et al., supra; DNA Cloning, vols I & II, supra;Nucleic Acid Hybridization, supra.

A sequence “functionally equivalent” to a Mda-5 promoter sequence is onewhich functions in the same manner as the Mda-5 promoter sequence. Thus,a promoter sequence “functionally equivalent” to the Mda-5 promoterdescribed herein is one which is capable of directing transcription of adownstream coding sequence in substantially similar timeframes ofexpression and in substantially similar amounts and with substantiallysimilar tissue specificity as the Mda-5 promoter.

In general terms, an “analog” is understood to be a functionalequivalent of a given substance and can be a substitute for saidsubstance, including as a therapeutic substitute. An analog also can bea structural equivalent. As used herein, a “Mda-5 analog” is a substancethat mimics a biological effect induced and/or mediated by Mda-5. Anysubstance having such mimetic properties, regardless of the chemical orbiochemical nature thereof, can be used as a Mda-5 analog herein. Asused herein, an Mda-5 analog can be referred to as a “mimic” or a“mimetic”.

A DNA “coding sequence” or a “nucleotide sequence encoding” a particularprotein, is a DNA sequence which is transcribed and translated into apolypeptide in vivo or in vitro when placed under the control ofappropriate regulatory sequences.

The boundaries of the coding sequence are determined by a start codon atthe 5′-(amino) terminus and a translation stop codon at the 3′-(carboxy)terminus. A coding sequence can include, but is not limited to,procaryotic sequences, cDNA from eucaryotic mRNA, genomic DNA sequencesfrom eucaryotic (e.g., mammalian) sources, viral RNA or DNA, and evensynthetic nucleotide sequences. A transcription termination sequencewill usually be located 3′ to the coding sequence.

DNA “control sequences” refers collectively to promoter sequences,polyadenylation signals, transcription termination sequences, upstreamregulatory domains, enhancers, and the like, untranslated regions,including 5′-UTRs and 3′-UTRs, which collectively provide for thetranscription and translation of a coding sequence in a host cell.

“Operably linked” refers to an arrangement of nucleotide sequenceelements wherein the components so described are configured so as toperform their usual function. Thus, control sequences operably linked toa coding sequence are capable of effecting the expression of the codingsequence. The control sequences need not be contiguous with the codingsequence, so long as they function to direct the expression thereof.Thus, for example, intervening untranslated yet transcribed sequencescan be present between a promoter sequence and the coding sequence andthe promoter sequence can still be considered “operably linked” to thecoding sequence.

A control sequence “directs the transcription” of a coding sequence in acell when RNA polymerase will bind the promoter sequence and transcribethe coding sequence into mRNA, which is then translated into thepolypeptide encoded by the coding sequence.

A cell has been “transformed” by exogenous DNA when such exogenous DNAhas been introduced inside the cell membrane. Exogenous DNA may or maynot be integrated (covalently linked) into chromosomal DNA making up thegenome of the cell. In procaryotes and yeasts, for example, theexogenous DNA may be maintained on an episomal element, such as aplasmid. In eucaryotic cells, a stably transformed cell is generally onein which the exogenous DNA has become integrated into the chromosome sothat it is inherited by daughter cells through chromosome replication,or one which includes stably maintained extrachromosomal plasmids. Thisstability is demonstrated by the ability of the eucaryotic cell toestablish cell lines or clones comprised of a population of daughtercells containing the exogenous DNA.

A “heterologous” region of a DNA construct is an identifiable segment ofDNA within or attached to another DNA molecule that is not found inassociation with the other molecule in nature. For example, a sequenceencoding a protein other than an Mda-5 is considered a heterologoussequence when linked to an Mda-5 promoter. Similarly, a sequenceencoding an Mda gene (i.e., Mda-6, Mda-7) will be consideredheterologous when linked to an Mda gene promoter with which it is notnormally associated. Another example of a heterologous coding sequenceis a construct where the coding sequence itself is not found in nature(e.g., synthetic sequences having codons different from the nativegene). Likewise, a chimeric sequence, comprising a heterologousstructural gene and a gene encoding an Mda or a portion of an Mda,linked to an Mda promoter, whether derived from the same or a differentMda gene, will be considered heterologous since such chimeric constructsare not normally found in nature. Allelic variation or naturallyoccurring mutational events do not give rise to a heterologous region ofDNA, as used herein.

Vectors

Especially preferred are virus based vectors. In the case of eukaryoticcells, retrovirus or adenovirus based vectors are preferred. Suchvectors contain all or a part of a viral genome, such as long termrepeats (“LTRs”), promoters (e.g., CMV promoters, SV40 promoter, RSVpromoter), enhancers, and so forth. When the host cell is a prokaryote,bacterial viruses, or phages, are preferred. Exemplary of such vectorsare vectors based upon, e.g., lambda phage. In any case, the vector maycomprise elements of more than one virus.

The resulting vectors are transfected or transformed into a host cell,which may be eukaryotic or prokaryotic.

The gene transfer vector of the present invention may additionallycomprise a gene encoding a marker or reporter molecule to more easilytrace expression of the vector.

Examples of such reporter molecules which can be employed in the presentinvention are well-known in the art and include beta-galactosidase(Fowler et al, Proc. Natl. Acad. Sci., USA, 74:1507 (1977)), luciferase(Tu et al, Biochem., 14:1970 (1975)), and chloramphenicolacetyltransferase (Gorman et al, Mol. Cell. Biol., 2:1044-1051 (1982)).

The gene transfer vector may contain more than one gene encoding thesame or different foreign polypeptides or RNAS.

The gene transfer vector may be any construct which is able to replicatewithin a host cell and includes plasmids, DNA viruses, retroviruses, aswell as isolated nucleotide molecules. Liposome-mediated transfer of thegene transfer vector may also be carried out in the present invention.

Examples of such plasmids which can be employed in the present inventioninclude pGL3-based plasmids (Promega). An example of such DNA viruseswhich can be employed in the present invention are adenoviruses.

Adenoviruses have attracted increasing attention as expression vectors,especially for human gene therapy (Berkner, Curr. Top. Microbiol.Immunol., 158:39-66 (1992)).

Examples of such adenovirus serotypes which can be employed in thepresent invention are well-known in the art and include more than 40different human adenoviruses, e.g., Ad12 (subgenus A), Ad3 and Ad7(Subgenus B), Ad2 and Ad5 (Subgenus C), Ad8 (Subgenus D), Ad4 (SubgenusE), Ad40 (Subgenus F) (Wigand et al, In: Adenovirus DNA, Doerfler, Ed.,Martinus Nijhoff Publishing, Boston, pp. 408-441 (1986)). Ad5 ofsubgroup C is the preferred adenovirus employed in the presentinvention. This is because Ad5 is a human adenovirus about which a greatdeal of biochemical and genetic information is known, and it hashistorically been used for most constructions employing adenovirus as avector. Also, adenoviral vectors are commercially available, e.g., pCA3(Microbix Biosystems Inc.).

Methods for producing adenovirus vectors are well-known in the art(Berkner et al, Nucleic Acids Res., 11:6003-6020 (1983); van Doren etal, Mol. Cell. Biol., 4:1653-1656 (1984); Ghosh-Choudhury et al,Biochem. Biophys. Res. Commun., 147:964-973 (1987); McGrory et al,Virol., 163:614-617 (1988); and Gluzman et al, In: Eurkaryotic ViralVectors, Ed. Gluzman, Y. pages 187-192, Cold Spring Harbor Laboratory(1982)).

Functionally Equivalent

Nucleic acid molecules which are “functionally equivalent” to Mda-5promoter or Mda-5 cDNA retain the functional properties of the Mda-5cDNA or MDA-5 promoter. The nucleic acid molecule may be a derivative ofthe Mda-5 cDNA or promoter such that there are substitutions, deletions,insertions or alterations in the nucleotide sequence which do not altersubstantially the function of the nucleic acid. For example, a promotermolecule which is a functional equivalent of Mda-5 promoter having suchsubstitutions will still permit the tissue specific expression of a geneof interest operably linked thereto and expressed in an organism.Modification is permitted so long as the derivative molecules retain itsincreased potency compared to Mda-5 promoter alone and its tissuespecificity. A functional equivalent of Mda-5 cDNA will encode a proteinwhich retains substantially the same biological functions which arecharacteristic of Mda-5.

The promoter of the present invention in one embodiment is operablylinked to a gene of interest. Such a gene of interest is preferably atherapeutic gene. Examples of therapeutic genes include suicide genes,envisioned for the treatment of cancer, for example. These are genessequences the expression of which produces a protein or agent thatinhibits tumor cell growth or induces tumor cell death. Suicide genesinclude genes encoding enzymes, oncogenes, tumor suppressor genes, genesencoding toxins, genes encoding cytokines, or a gene encodingoncostatin. The purpose of the therapeutic gene is to inhibit the growthof or kill cancer cells or produce cytokines or other cytotoxic agentswhich directly or indirectly inhibit the growth of or kill the cancercell.

Suitable enzymes include thymidine kinase (TK), xanthine-guaninephosphoribosyltransferase (GPT) gene from E. coli or E. coli cytosinedeaminase (CD), or hypoxanthine phosphoribosyl transferase (HPRT).

Suitable oncogenes and tumor suppressor genes include neu, EGF, ras(including H, K, and N ras), p53, Retinoblastoma tumor suppressor gene(Rb), Wilm's Tumor Gene Product, Phosphotyrosine Phosphatase (PTPase),and nm23. Suitable toxins include Pseudomonas exotoxin A and S;diphtheria toxin (DT); E. coli LT toxins, Shiga toxin, Shiga-like toxins(SLT-1, -2), ricin, abrin, supporin, and gelonin.

In one embodiment, the gene of interest is a cytokine. Suitablecytokines include interferons, GM-CSF interleukins, tumor necrosisfactor (TNF) (Wong G, et al., Human GM-CSF: Molecular cloning of thecomplementary DNA and purification of the natural and recombinantproteins. Science 1985; 228:810); WO9323034 (1993); Horisberger M. A.,et al., Cloning and sequence analyses of cDNAs for interferon-beta andvirus-induced human Mx proteins reveal that they contain putativeguanine nucleotide-binding sites: functional study of the correspondinggene promoter. Journal of Virology, 1990 March, 64(3):1171-81; Li Y P etal., Proinflammatory cytokines tumor necrosis factor-alpha and IL-6, butnot IL-1, down-regulate the osteocalcin gene promoter. Journal ofImmunology, Feb. 1, 1992, 148(3):788-94; Pizarro T. T., et al. Inductionof TNF alpha and TNF beta gene expression in rat cardiac transplantsduring allograft rejection. Transplantation, 1993 August,56(2):399-404). (Breviario F., et al., Interleukin-1-inducible genes inendothelial cells. Cloning of a new gene related to C-reactive proteinand serum amyloid P component. Journal of Biological Chemistry, Nov. 5,1992, 267(31):22190-7; Espinoza-Delgado I., et al., Regulation of IL-2receptor subunit genes in human monocytes. Differential effects of IL-2and IFN-gamma. Journal of Immunology, Nov. 1, 1992, 149(9):2961-8;Algate P. A., et al., Regulation of the interleukin-3 (IL-3) receptor byIL-3 in the fetal liver-derived FL5.12 cell line. Blood, 1994 May 1,83(9):2459-68; Cluitmans F. H., et al., IL-4 down-regulates IL-2-,IL-3-, and GM-CSF-induced cytokine gene expression in peripheral bloodmonocytes. Annals of Hematology, 1994 June, 68(6):293-8; Lagoo, A. S.,et al., IL-2, IL-4, and IFN-gamma gene expression versus secretion insuperantigen-activated T cells. Distinct requirement for costimulatorysignals through adhesion molecules. Journal of Immunology, Feb. 15,1994, 152(4):1641-52; Martinez O. M., et al., IL-2 and IL-5 geneexpression in response to alloantigen in liver allograft recipients andin vitro. Transplantation, 1993 May, 55(5):1159-66; Pang G, et al.,GM-CSF, IL-1 alpha, IL-1 beta, IL-6, IL-8, IL-10, ICAM-1 and VCAM-1 geneexpression and cytokine production in human duodenal fibroblastsstimulated with lipopolysaccharide, IL-1 alpha and TNF-alpha. Clinicaland Experimental Immunology, 1994 June, 96(3):437-43; Ulich T. R., etal., Endotoxin-induced cytokine gene expression in vivo. III. IL-6 mRNAand serum protein expression and the in vivo hematologic effects ofIL-6. Journal of Immunology, Apr. 1, 1991, 146(7):2316-23; Mauviel A.,et al., Leukoregulin, a T cell-derived cytokine, induces IL-8 geneexpression and secretion in human skin fibroblasts. Demonstration andsecretion in human skin fibroblasts. Demonstration of enhanced NF-kappaB binding and NF-kappa B-driven promoter activity. Journal ofImmunology, Nov. 1, 1992, 149(9):2969-76).

The gene of interest is a growth factor in one embodiment. Growthfactors include Transforming Growth Factor-alpha (TGF-alpha) and beta(TGF-beta), cytokine colony stimulating factors (Shimane M., et al.,Molecular cloning and characterization of G-CSF induced gene cDNA.Biochemical and Biophysical Research Communications, Feb. 28, 1994,199(1):26-32; Kay A. B., et al., Messenger RNA expression of thecytokine gene cluster, interleukin 3 (IL-3), IL-4, IL-5, andgranulocyte/macrophage colony-stimulating factor, in allergen-inducedlate-phase cutaneous reactions in atopic subjects. Journal ofExperimental Medicine, Mar. 1, 1991, 173(3):775-8; de Wit H, et al.,Differential regulation of M-CSF and IL-6 gene expression in monocyticcells. British Journal of Haematology, 1994 February, 86(2):259-64;Sprecher E., et al., Detection of IL-1 beta, TNF-alpha, and IL-6 genetranscription by the polymerase chain reaction in keratinocytes,Langerhans cells and peritoneal exudate cells during infection withherpes simplex virus-1. Archives of Virology, 1992, 126(1-4):253-69).

Preferred vectors for use in the methods of the present invention areviral including adenoviruses, retroviral, vectors, adeno-associatedviral (AAV) vectors.

The viral vector selected should meet the following criteria: 1) thevector must be able to infect the tumor cells and thus viral vectorshaving an appropriate host range must be selected; 2) the transferredgene should be capable of persisting and being expressed in a cell foran extended period of time; and 3) the vector should be safe to the hostand cause minimal cell transformation. Retroviral vectors andadenoviruses offer an efficient, useful, and presently thebest-characterized means of introducing and expressing foreign genesefficiently in mammalian cells. These vectors have very broad host andcell type ranges, express genes stably and efficiently. The safety ofthese vectors has been proved by many research groups. In fact many arein clinical trials.

Other virus vectors that may be used for gene transfer into cells forcorrection of disorders include retroviruses such as Moloney murineleukemia virus (MoMuLV); papovaviruses such as JC, SV40, polyoma,adenoviruses; Epstein-Barr Virus (EBV); papilloma viruses, e.g. bovinepapilloma virus type I (BPV); vaccinia and poliovirus and other humanand animal viruses.

Adenoviruses have several properties that make them attractive ascloning vehicles (Bachettis et al.: Transfer of gene for thymidinekinase-deficient human cells by purified herpes simplex viral DNA. PNASUSA, 1977 74:1590; Berkner, K. L.: Development of adenovirus vectors forexpression of heterologous genes. Biotechniques, 1988 6:616;Ghosh-Choudhury G., et al., Human adenovirus cloning vectors based oninfectious bacterial plasmids. Gene 1986; 50:161; Hag-Ahmand Y., et al.,Development of a helper-independent human adenovirus vector and its usein the transfer of the herpes simplex virus thymidine kinase gene. JVirol 1986; 57:257; Rosenfeld M., et al., Adenovirus-mediated transferof a recombinant alpha.sub.1-antitrypsin gene to the lung epithelium invivo. Science 1991; 252:431).

For example, adenoviruses possess an intermediate sized genome thatreplicates in cellular nuclei; many serotypes are clinically innocuous;adenovirus genomes appear to be stable despite insertion of foreigngenes; foreign genes appear to be maintained without loss orrearrangement; and adenoviruses can be used as high level transientexpression vectors with an expression period up to 4 weeks to severalmonths. Extensive biochemical and genetic studies suggest that it ispossible to substitute up to 7-7.5 kb of heterologous sequences fornative adenovirus sequences generating viable, conditional,helper-independent vectors (Kaufman R. J.; identification of thecomponent necessary for adenovirus translational control and theirutilization in cDNA expression vectors. PNAS USA, 1985 82:689).

AAV is a small human parvovirus with a single stranded DNA genome ofapproximately 5 kb. This virus can be propagated as an integratedprovirus in several human cell types. AAV vectors have several advantagefor human gene therapy. For example, they are trophic for human cellsbut can also infect other mammalian cells; (2) no disease has beenassociated with AAV in humans or other animals; (3) integrated AAVgenomes appear stable in their host cells; (4) there is no evidence thatintegration of AAV alters expression of host genes or promoters orpromotes their rearrangement; (5) introduced genes can be rescued fromthe host cell by infection with a helper virus such as adenovirus.

HSV-1 vector system facilitates introduction of virtually any gene intonon-mitotic cells (Geller et al. an efficient deletion mutant packagingsystem for a defective herpes simplex virus vectors: Potentialapplications to human gene therapy and neuronal physiology. PNAS USA,1990 87:8950).

Another vector for mammalian gene transfer is the bovine papillomavirus-based vector (Sarver N, et al., Bovine papilloma virus DNA: Anovel eukaryotic cloning vector. Mol Cell Biol 1981; 1:486).

Vaccinia and other poxvirus-based vectors provide a mammalian genetransfer system. Vaccinia virus is a large double-stranded DNA virus of120 kilodaltons (kd) genomic size (Panicali D, et al., Construction ofpoxvirus as cloning vectors: Insertion of the thymidine kinase gene fromherpes simplex virus into the DNA of infectious vaccine virus. Proc NatlAcad Sci USA 1982; 79:4927; Smith et al. infectious vaccinia virusrecombinants that express hepatitis B virus surface antigens. Nature,1983 302:490.)

Retroviruses are packages designed to insert viral genes into host cells(Guild B, et al., Development of retrovirus vectors useful forexpressing genes in cultured murine embryonic cells and hematopoieticcells in vivo. J Virol 1988; 62:795; Hock R. A., et al., Retrovirusmediated transfer and expression of drug resistance genes in humanhemopoietic progenitor cells. Nature 1986; 320:275).

The basic retrovirus consists of two identical strands of RNA packagedin a proviral protein. The core surrounded by a protective coat calledthe envelope, which is derived from the membrane of the previous hostbut modified with glycoproteins contributed by the virus.

Markers and amplifiers can also be employed in the gene transfer vectorsof the invention. A variety of markers are known which are useful inselecting for transformed cell lines and generally comprise a gene whoseexpression confers a selectable phenotype on transformed cells when thecells are grown in an appropriate selective medium. Such markers formammalian cell lines include, for example, the bacterialxanthine-guanine phosphoribosyl transferase gene, which can be selectedfor in medium containing mycophenolic acid and xanthine (Mulligan et al.(1981) Proc. Natl. Acad. Sci. USA 78:2072-2076), and the aminoglycosidephosphotransferase gene (specifying a protein that inactivates theantibacterial action of neomycin/kanamycin derivatives), which can beselected for using medium containing neomycin derivatives such as G418which are normally toxic to mammalian cells (Colbere-Garapin et al.(1981) J. Mol. Biol. 150:1-14). Useful markers for other eucaryoticexpression systems, are well known to those of skill in the art.

Infection of cells can be carried out in vitro or in vivo. In vitroinfection of cells is performed by adding the gene transfer vectors tothe cell culture medium. When infection is carried out in vivo, thesolution containing the gene transfer vectors may be administered by avariety of modes, depending on the tissue which is to be infected.Examples of such modes of administration include injection of genetransfer vectors into the skin, topical application onto the skin,direct application to a surface of epithelium, or instillation into anorgan (e.g., time release patch or capsule below the skin or into atumor), oral administration, injection into the cerebro-spinal fluid,intranasal application, application into eye by dropper, etc.

Expression can be amplified by placing an amplifiable gene, such as themouse dihydrofolate reductase (dhfr) gene adjacent to the codingsequence. Cells can then be selected for methotrexate resistance indhfr-deficient cells. See, e.g. Urlaub et al. (1980) Proc. Natl. Acad.Sci. USA 77:4216-4220; Rungold et al. (1981) J. Mol. and Appl. Genet.1:165-175.

The above-described system can be used to direct the expression of awide variety of procaryotic, eucaryotic and viral proteins, (genes ofinterest) including, for example, viral glycoproteins suitable for useas vaccine antigens, immunomodulators for regulation of the immuneresponse, hormones, cytokines and growth factors, as well as proteinsuseful in the production of other biopharmaceuticals.

It may also be desirable to produce mutants or analogs of the proteinsof interest. See description of “functionally equivalent” nucleic acidshereinabove. Such mutants or analogs of the proteins of interest in oneembodiment are expressed from functionally equivalent nucleic acids ofthe gene of interest or of Mda-5 cDNA. Mutants or analogs may beprepared by the deletion of a portion of the sequence encoding theprotein, by insertion of a sequence, and/or by substitution of one ormore nucleotides within the sequence. Techniques for modifyingnucleotide sequences, such as site-directed mutagenesis, are well knownto those skilled in the art. See, e.g., Sambrook et al., supra; DNACloning, Vols. I and II, supra; Nucleic Acid Hybridization, supra.

For purposes of the present invention, it may be desirable to furtherengineer the coding sequence to effect secretion of the polypeptide fromthe host organism. This enhances clone stability and prevents the toxicbuild up of proteins in the host cell so that expression can proceedmore efficiently. Homologous signal sequences can be used for thispurpose with proteins normally found in association with a signalsequence. Additionally, heterologous leader sequences which provide forsecretion of the protein can be added to the constructs. Preferably,processing sites will be included such that the leader fragment can becleaved from the protein expressed therewith. (See, e.g., U.S. Pat. No.4,336,246 for a discussion of how such cleavage sites can beintroduced). The leader sequence fragment typically encodes a signalpeptide comprised of hydrophobic amino acids.

In one embodiment of the invention, a heterologous gene sequence, i.e.,a therapeutic gene, is inserted into the nucleic acid molecule of theinvention. Other embodiments of the isolated nucleic acid molecule ofthe invention include the addition of a single enhancer element ormultiple enhancer elements which amplify the expression of theheterologous therapeutic gene without compromising tissue specificity.

The transformation procedure used depends upon the host to betransformed. Mammalian cells can conveniently be transformed using, forexample, DEAE-dextran based procedures, calcium phosphate precipitation(Graham, F. L. and Van der Eb, A. J. (1973) Virology 52:456-467),protoplast fusion, liposome-mediated transfer, polybrene-mediatedtransfection and direct microinjection of the DNA into nuclei. Bacterialcells will generally be transformed using calcium chloride, either aloneor in combination with other divalent cations and DMSO (Sambrook,Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, SecondEdition (1989)). DNA can also be introduced into bacterial cells byelectroporation. Methods of introducing exogenous DNA into yeast hoststypically include either the transformation of spheroplasts ortransformation of intact yeast cells treated with alkali cations.

The constructs can also be used in gene therapy or nucleic acidimmunization, to direct the production of the desired gene product invivo, by administering the expression constructs directly to a subjectfor the in vivo translation thereof. See, e.g. EPA Publication No.336,523 (Dreano et al., published Oct. 11, 1989). Alternatively, genetransfer can be accomplished by transfecting the subject's cells ortissues with the expression constructs ex vivo and reintroducing thetransformed material into the host. The constructs can be directlyintroduced into the host organism, i.e., by injection (see InternationalPublication No. WO/90/11092; and Wolff et al., (1990) Science247:1465-1468). Liposome-mediated gene transfer can also be accomplishedusing known methods. See, e.g., Hazinski et al., (1991) Am. J. Respir.Cell Mol. Biol. 4:206-209; Brigham et al. (1989) Am. J. Med. Sci.298:278-281; Canonico et al. (1991) Clin. Res. 39:219 A; and Nabel etal. (1990) Science 249:1285-1288. Targeting agents, such as antibodiesdirected against surface antigens expressed on specific cell types, canbe covalently conjugated to the liposomal surface so that the nucleicacid can be delivered to specific tissues and cells for localadministration.

Human Gene Therapy and Diagnostic Use of Vector

There are several protocols for human gene therapy which have beenapproved for use by the Recombinant DNA Advisory Committee (RAC) whichconform to a general protocol of target cell infection andadministration of transfected cells (see for example, Blaese, R. M., etal., 1990; Anderson, W. F., 1992; Culver, K. W. et al., 1991). Inaddition, U.S. Pat. No. 5,399,346 (Anderson, W. F. et al., Mar. 21,1995, U.S. Serial No. 220,175) describes procedures for retroviral genetransfer. The contents of these support references are incorporated intheir entirety into the subject application. Retroviral-mediated genetransfer requires target cells which are undergoing cell division inorder to achieve stable integration hence, cells are collected from asubject often by removing blood or bone marrow. It may be necessary toselect for a particular subpopulation of the originally harvested cellsfor use in the infection protocol. Then, a retroviral vector containingthe gene(s) of interest would be mixed into the culture medium. Thevector binds to the surface of the subject's cells, enters the cells andinserts the gene of interest randomly into a chromosome. The gene ofinterest is now stably integrated and will remain in place and be passedto all of the daughter cells as the cells grow in number. The cells maybe expanded in culture for a total of 9-10 days before reinfusion(Culver et al., 1991). As the length of time the target cells are leftin culture increases, the possibility of contamination also increases,therefore a shorter protocol would be more beneficial.

This invention provides for the construction of retrovirus vectorscontaining the Mda-5 cDNA in a replicable gene transfer vector or Mda-5promoter linked to a gene of interest for use in gene therapy or fordiagonistic uses. The efficiency of transduction of these vectors can betested in cell culture systems.

Uses of the Compositions of the Invention

This invention involves targeting a gene-of-interest to the a cancercell so that the protein encoded by the gene is expressed and directlyor indirectly ameliorate the diseased state.

After infecting a susceptible cell, the transgene driven by a specificpromoter in the vector expresses the protein encoded by the gene. Theuse of the highly specific gene vector will allow selective expressionof the specific genes in cancer cells.

In one embodiment, the present invention relates to a process foradministering modified vectors into the skin to treat skin cancer ordisorders associated with the skin. More particularly, the inventionrelates to the use of vectors carrying functional therapeutic genes toproduce molecules that are capable of directly or indirectly affectingcells in the skin to repair damage sustained by the cells from defects,disease or trauma.

Preferably, for treating cancer or for treating defects, disease ordamage of cells in the skin, vectors of the invention include atherapeutic gene or transgenes, for example a gene encoding TK. Thegenetically modified vectors are administered into the skin to treatdefects, disease such as skin cancer by introducing a therapeutic geneproduct or products into the skin that enhance the production ofendogenous molecules that have ameliorative effects in vivo.

The basic tasks in the present method of the invention are isolating thegene of interest, selecting the proper vector vehicle to deliver thegene of interest to the body, administering the vector having the geneof interest into the body, and achieving appropriate expression of thegene of interest. The present invention provides packaging the clonedgenes, i.e. the genes of interest, in such a way that they can beinjected directly into the bloodstream or relevant organs of patientswho need them. The packaging will protect the foreign DNA fromelimination by the immune system and direct it to appropriate tissues orcells.

In one embodiment of the invention, the gene of interest (desired codingsequence) is a tumor suppressor gene. The tumor suppressor gene may bep21, RB (retinoblastoma) or p53. One of skill in the art would know ofother tumor suppressor genes, Recent U.S. Pat. Nos. 6,025,127 and5,912,236 are hereby incorporated by reference to more explicitlydescribe the state of the art as to tumor suppressor genes.

Along with the human or animal gene of interest another gene, e.g., aselectable marker, can be inserted that will allow easy identificationof cells that have incorporated the modified retrovirus. The criticalfocus on the process of gene therapy is that the new gene must beexpressed in target cells at an appropriate level with a satisfactoryduration of expression.

The methods described below to modify vectors and administering suchmodified vectors into the skin are merely for purposes of illustrationand are typical of those that might be used. However, other proceduresmay also be employed, as is understood in the art.

Most of the techniques used to construct vectors and the like are widelypracticed in the art, and most practitioners are familiar with thestandard resource materials which describe specific conditions andprocedures. However, for convenience, the following paragraphs may serveas a guideline.

General Methods for Vector Construction

Construction of suitable vectors containing the desired therapeutic genecoding and control sequences employs standard ligation and restrictiontechniques, which are well understood in the art (see Maniatis et al.,in Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, New York (1982)). Isolated plasmids, DNA sequences, orsynthesized oligonucleotides are cleaved, tailored, and religated in theform desired.

Site-specific DNA cleavage is performed by treating with the suitablerestriction enzyme (or enzymes) under conditions which are generallyunderstood in the art, and the particulars of which are specified by themanufacturer of these commercially available restriction enzymes (See,e.g. New England Biolabs Product Catalog). In general, about 1 μg ofplasmid or DNA sequences is cleaved by one unit of enzyme in about 20 μlof buffer solution. Typically, an excess of restriction enzyme is usedto insure complete digestion of the DNA substrate.

Incubation times of about one hour to two hours at about 37 degree. C.are workable, although variations can be tolerated. After eachincubation, protein is removed by extraction with phenol/chloroform, andmay be followed by ether extraction, and the nucleic acid recovered fromaqueous fractions by precipitation with ethanol. If desired, sizeseparation of the cleaved fragments may be performed by polyacrylamidegel or agarose gel electrophoresis using standard techniques. A generaldescription of size separations is found in Methods in Enzymology65:499-560 (1980).

Restriction cleaved fragments may be blunt ended by treating with thelarge fragment of E. coli DNA polymerase I (Klenow) in the presence ofthe four deoxynucleotide triphosphates (dNTPs) using incubation times ofabout 15 to 25 min at 20.degree. C. to 25.degree. C. in 50 mM Tris (pH7.6) 50 mM NaCl, 6 mM MgCl.sub.2, 6 mM DTT and 5-10.mu.M dNTPs. TheKlenow fragment fills in at 5′ sticky ends but chews back protruding 3′single strands, even though the four dNTPs are present. If desired,selective repair can be performed by supplying only one of the dNTPs, orwith selected dNTPs, within the limitations dictated by the nature ofthe sticky ends. After treatment with Klenow, the mixture is extractedwith phenol/chloroform and ethanol precipitated. Treatment underappropriate conditions with S1 nuclease or Bal-31 results in hydrolysisof any single-stranded portion.

Ligations are performed in 10-50 μl volumes under the following standardconditions and temperatures using T4 DNA ligase. Ligation protocols arestandard (D. Goeddel (ed.) Gene Expression Technology: Methods inEnzymology (1991)). In vector construction employing “vector fragments”,the vector fragment is commonly treated with bacterial alkalinephosphatase (BAP) or calf intestinal alkaline phosphatase (CIP) in orderto remove the 5′ phosphate and prevent religation of the vector.Alternatively, religation can be prevented in vectors which have beendouble digested by additional restriction enzyme digestion of theunwanted fragments.

Suitable vectors include viral vector systems e.g. ADV, RV, and AAV (R.J. Kaufman “Vectors used for expression in mammalian cells” in GeneExpression Technology, edited by D. V. Goeddel (1991).

Many methods for inserting functional DNA transgenes into cells areknown in the art. For example, non-vector methods include nonviralphysical transfection of DNA into cells; for example, microinjection(DePamphilis et al., BioTechnique 6:662-680 (1988)); liposomal mediatedtransfection (Felgner et al., Proc. Natl. Acad. Sci. USA, 84:7413-7417(1987), Feigner and Holm, Focus 11:21-25 (1989) and Felgner et al.,Proc. West. Pharmacol. Soc. 32: 115-121 (1989)) and other methods knownin the art.

Administration of Modified Vectors Into Subject

One way to get DNA into a target cell is to put it inside a membranebound sac or vesicle such as a spheroplast or liposome, or by calciumphosphate precipitation (CaPO.sub.4) (Graham F. and Van der Eb, A.,Virology 52:456 1973; Schaefer-Ridder M., et al., Liposomes as genecarriers: Efficient transduction of mouse L cells by thymidine kinasegene. Science 1982; 215:166; Stpyridis J. C., et al., Construction oftransferrin-coated liposomes for in vivo transport of exogenous DNA tobone marrow erythroblasts in rabbits. Exp Cell Res 1986; 164:568-572).

A vesicle can be constructed in such a way that its membrane will fusewith the outer membrane of a target cell. The vector of the invention invesicles can home into the cancer cells.

The spheroplasts are maintained in high ionic strength buffer until theycan be fused through the mammalian target cell using fusogens such aspolyethylene glycol.

Liposomes are artificial phospholipid vesicles. Vesicles range in sizefrom 0.2 to 4.0 micrometers and can entrap 10% to 40% of an aqueousbuffer containing macromolecules. The liposomes protect the DNA fromnucleases and facilitate its introduction into target cells.Transfection can also occur through electroporation.

Before administration, the modified vectors are suspended in completePBS at a selected density for injection. In addition to PBS, anyosmotically balanced solution which is physiologically compatible withthe subject may be used to suspend and inject the modified vectors intothe host.

For injection, the cell suspension is drawn up into the syringe andadministered to anesthetized recipients. Multiple injections may be madeusing this procedure. The viral suspension procedure thus permitsadministration of genetically modified vectors to any predetermined sitein the skin, is relatively non-traumatic, allows multipleadministrations simultaneously in several different sites or the samesite using the same viral suspension. Multiple injections may consist ofa mixture of therapeutic genes.

Survival of the Modified Vectors So Administered

Expression of a gene is controlled at the transcription, translation orpost-translation levels. Transcription initiation is an early andcritical event in gene expression. This depends on the promoter andenhancer sequences and is influenced by specific cellular factors thatinteract with these sequences. The transcriptional unit of manyprokaryotic genes consists of the promoter and in some cases enhancer orregulator elements (Banerji et al., Cell 27:299 (1981); Corden et al.,Science 209:1406 (1980); and Breathnach and Chambon, Ann. Rev. Biochem.50:349 (1981)).

For retroviruses, control elements involved in the replication of theretroviral genome reside in the long terminal repeat (LTR) (Weiss etal., eds., In: The molecular biology of tumor viruses: RNA tumorviruses, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.(1982)).

Moloney murine leukemia virus (MLV) and Rous sarcoma virus (RSV) LTRscontain promoter and enhancer sequences (Jolly et al., Nucleic AcidsRes. 11:1855 (1983); Capecchi et al., In: Enhancer and eukaryotic geneexpression, Gulzman and Shenk, eds., pp. 101-102, Cold Spring HarborLaboratories, Cold Spring Harbor, N.Y.).

Promoter and enhancer regions of a number of non-viral promoters havealso been described (Schmidt et al., Nature 314:285 (1985); Rossi and deCrombrugghe, Proc. Natl. Acad. Sci. USA 84:5590-5594 (1987)).

The present invention provides methods for maintaining and increasingexpression of therapeutic genes using a tissue specific promoter.

In addition to using viral and non-viral promoters to drive therapeuticgene expression, an enhancer sequence may be used to increase the levelof therapeutic gene expression. Enhancers can increase thetranscriptional activity not only of their native gene but also of someforeign genes (Armelor, Proc. Natl. Acad. Sci. USA 70:2702 (1973)).

For example, in the present invention, CMV enhancer sequences are usedwith the Mda-5 promoter to increase therapeutic gene expression.Therapeutic gene expression may also be increased for long term stableexpression after injection using cytokines to modulate promoteractivity.

The methods of the invention are exemplified by preferred embodiments inwhich modified vectors carrying a therapeutic gene are injectedintracerebrally into a subject.

The most effective mode of administration and dosage regimen for themolecules of the present invention depends upon the exact location ofthe melanoma being treated, the severity and course of the cancer, thesubject's health and response to treatment and the judgment of thetreating physician. Accordingly, the dosages of the molecules should betitrated to the individual subject. The molecules may be delivereddirectly or indirectly via another cell, autologous cells are preferred,but heterologous cells are encompassed within the scope of theinvention.

The interrelationship of dosages for animals of various sizes andspecies and humans based on mg/m.sup.2 of surface area is described byFreireich, E. J., et al. Cancer Chemother., Rep. 50 (4):219-244 (1966).Adjustments in the dosage regimen may be made to optimize the tumor cellgrowth inhibiting and killing response, e.g., doses may be divided andadministered on a daily basis or the dose reduced proportionallydepending upon the situation (e.g., several divided dose may beadministered daily or proportionally reduced depending on the specifictherapeutic situation).

It would be clear that the dose of the molecules of the inventionrequired to achieve cures may be further reduced with scheduleoptimization.

ADVANTAGES OF THE INVENTION

The Mda-5 promoter of the invention exhibits melanocyte tissuespecificity. Since the Mda-5 promoter of the invention istissue-specific it can only be activated in the targeted tissue, i.e.,the skin. Therefore, the genes of interest driven by the Mda-5 promoterwill be differentially expressed in these cells, minimizing systemictoxicity.

This invention is illustrated in the Experimental Details section whichfollows. These sections are set forth to aid in an understanding of theinvention but are not intended to, and should not be construed to, limitin any way the invention as set forth in the claims which followthereafter.

EXPERIMENTAL DETAILS Example 1 Melanoma Differentiation AssociatedGene-5, Mda-5, a Novel Interferon Inducible Gene with StructuralSimilarities to RNA Helicases and CARD Motif Containing Proteins

Melanoma differentiation associated gene-5, mda-5, is induced duringterminal differentiation in human melanoma cells treated with thecombination of recombinant fibroblast interferon (IFN-) and theantileukemic compound mezerein (MEZ). The complete open reading frame ofthe mda-5 cDNA and its promoter region has now been identified andcharacterized. Mda-5 encodes a 116.7-kDa protein that contains a caspaserecruitment domain (CARD) and an RNA helicase motif. Treatment of HO-1human melanoma and human skin fibroblast cells with IFN-α, IFN-0, IFN-γ,TNF-α and poly IC induce mda-5 expression. IFN-γ and poly IC are themost potent single inducers of mda-5 expression, resulting in a ≧5-foldhigher induction than with other inducers. Induction of mda-5 expressionby IFN-β is also apparent in normal and tumor cell lines of diverseorigin. Thus, mda-5 is a novel IFN-β-responsive gene. MEZ, whichreversibly induces specific markers of differentiation in HO-1 cells,does not induce mda-5 expression, whereas it increases both the level ofsteady-state mda-5 mRNA and mda-5 RNA transcription. The finding thatmost organs, except brain and lung, contain low levels of mda-5transcripts suggest that the biological role of mda-5 may be closelyrelated to its induction by exogenous agents. Nuclear run-on assaysindicate that the level of regulation of mda-5 occurs transcriptionally.The half-life of mda-5 following treatment with IFN-β or IFN-β+MEZ isbetween 5-6 hr, confirming that the primary regulation of mda-5 by theseagents occurs by enhanced RNA transcription rates. Isolation andcharacterization of the promoter region of mda-5, provides furtherdocumentation that the primary mode of regulation of this gene involveschanges in RNA transcription. MDA-5 protein was detected at thepredicted size by in vitro translation and Western blot analysis oftransiently expressed fusion proteins. GFP-mda-5 fusion proteins wereproduced and found to localize in the cytoplasm where mda-5 may effectson mRNA translation, mRNA sequestration and decay of specific messages.Ectopic expression of mda-5 reduces the colony-forming efficiency ofHO-1 melanoma cells by ˜70%, which suggests a growth inhibitory or apro-apoptotic role of mda-5. In these contexts, mda-5 may play a keyrole in growth inhibition induced by IFN-β and may also function inapoptotic signaling.

Introduction

Abnormalities in differentiation are common occurrences in human cancers((i) Fisher and Grant, 1985; (2) Waxman, 1995). Moreover, as cancercells evolve, ultimately developing new phenotypes or acquiring afurther elaboration of preexisting transformation-related properties,the degree of expression of differentiation-associated traits oftenundergo a further decline. These observations have been exploited as anovel means of cancer therapy in which tumor cells are treated withagents that induce differentiation and a loss of cancerous properties, astrategy called differentiation therapy, ((2-4) Waxman et al., 1988,1991; Jiang et al., 1994; Waxman, 1995). In principle, differentiationtherapy may prove less toxic than currently employed chemotherapeuticapproaches, including radiation and treatment with toxic chemicals. Theability to develop rational schemes for applying differentiation therapyclinically require appropriate in vitro and in viva model systems foridentifying and characterizing the appropriate agent or agents that canmodulate differentiation in cancer cells without causing undue toxicityto normal cells.

Treatment of human melanoma cells with a combination of recombinanthuman fibroblast interferon (IFN-β) and the antileukemic compoundmezerein (MEZ) results in a rapid and irreversible suppression of growthand the induction of terminal cell differentiation ((5) Fisher et al.,1985). This process is associated with a number of changes in cellularphenotype and gene expression ((3, 6-7) Jiang et al., 1993, Jiang etal., 1994). To define the molecular basis of terminal differentiation inhuman melanoma cells subtraction hybridization has been employed ((8)Jiang and Fisher, 1993). In brief, cDNA libraries were prepared fromtemporal RNA samples obtained from HO-1 human melanoma cells treatedwith IFN-β+MEZ and control untreated HO-1 cells and control cDNAs weresubtracted away from differentiation-inducer treated cDNAs ((8) Jiangand Fisher, 1993). This approach resulted in an enrichment of genesdisplaying elevated expression as a function of treatment with thedifferent inducers and the induction of irreversible growth suppressionand terminal cell differentiation. Screening of the subtracteddifferentiation inducer treated HO-1 cDNA library identified both knownand novel cDNAs displaying elevated expression in differentiationinducer treated HO-1 cells ((3, 6, 8-14) Jiang and Fisher, 1993; Jianget al., 1994, 1995, 1996; Lin et al., 1994, 1996; Huang et al., 1999a,1999b). Four classes of genes, called melanoma differentiationassociated (mda) genes, have been cloned using this approach ((8) Jiangand Fisher, 1993). These include genes displaying elevated expression asa function of treatment with: IFN-β and IFN-β+MEZ (Type I mda genes);MEZ and IFN-β+MEZ (Type II mda genes); IFN-β, MEZ and IFN-β+MEZ (TypeIII mda genes); and predominantly with IFN-β+MEZ ((3,8) Jiang andFisher, 1993; Jiang et al., 1994). This approach has resulted in thecloning of both known and novel genes involved in important cellularprocesses, including cell cycle control (mda-6/p21), interferonsignaling (ISG-15, ISG-54), cancer growth control (mda-7), immuneinterferon response (mda-9), transcription control (c-jun, jun-B),immune recognition (HLA Class I) and cell membrane processes (α5integrin, βa integrin, fibronectin) ((3, 8-15) Jiang and Fisher, 1993;Jiang et al., 1994; Jiang et al., 1995a, 1995b, 1996, 1996; Lin et al.,1994, 1996).

Subtraction hybridization initially identified a small EST named mda-5.Expression of mda-5 was elevated in HO-1 cells treated with IFN-β+MEZand to a lesser extend by IFN β+IFN-γ. A complete mda-5 cDNA has nowbeen cloned and its properties determined. This gene is a novel earlyIFN responsive gene, whose activity is increased maximally by treatmentwith IFN-β and dsRNA. Moreover, the combination of IFN-β+MEZsynergistically induces mda-5 expression in HO-1 and additional celltypes, both normal and cancer. The protein structure of MDA-5 indicatespotential relationships to RNA helicases and genes containing CARDdomains. However, based on the structure of the MDA-5 protein this genemay represent a new member of the helicase gene family. Ectopicexpression of mda-5 induces growth suppression, as indicated by areduction in colony formation, in HO-1 human melanoma cells.Identification, cloning and analysis of upstream genomic sequences haveconfirmed that the mda-5 gene is responsive at a transcriptional toinduction primarily by IFN-β and dsRNA. A potential role for mda-5 ingrowth suppression induced by IFN and as a molecule involved in thecellular defense mechanism against viral infection is suggested.

Materials and Methods

Cell Culture and Treatment Protocol: HO-1 human melanoma cells, earlypassage human skin fibroblast (purchased from ATCC) and 293T cells weregrown in Dulbecco's modified Eagle's medium supplemented with 10% fetalbovine serum at 37° C. in a 5% CO2/95% air humidified incubator. Priorto treatment, cells were refed with fresh medium and exposed to theindicated compound(s) at the concentrations specified in the figurelegends.

Cloning and Sequencing of mda-5: The full length of the mda-5 cDNA wascloned by using the complete open reading frame (C-ORF) technology basedon the partial mda-5 EST sequence ((16) Kang and Fisher, 2000).Sequencing was performed by the dye-conjugated dideoxy chain terminationmethod. The ORF of mda-5 was cloned into the SmaI site of pcDEF3 inwhich mda-5 expression was regulated by the EF-1α promoter. Deletionmutant DN7 (D310-484 spanning the ATPase motif) was constructed byligation of BamHI-StuI fragment with Klenow-filled AlwNI-NotI fragmentinto pcDEF3. Antisense mda-5 expression vector was constructed bycloning the 1-1830 bp mda-5 cDNA fragment in an antisense orientationinto pcDEF3. GFP-mda-5 fusion expression vector was constructed byligation of the mda-5 cDNA into the SmaI site of pEGFP-C2. The sequencesof the expression vectors were verified as described above.

Northern Blot Analyses and Nuclear Run-On Assays: Total cellular RNAsamples were prepared by guanidium isothiocyanate/phenol extractionfollowed by isopropanol precipitation. Ten 10 μg of total RNA wereresolved in 1% agarose gels with formaldehyde and were transferred toNylon membranes. EcoRI fragment of mda-5 cDNA (2.5 kb) was labeled with32P using a multiprime labeling kit (Boehringer Mannheim) and used toprobe the transferred membrane. Nuclear run-on assays were performed aspreviously described ((17) Su et al., 1993, 1999). Probes used fornuclear run-on assays were prepared by RT-PCR and included the mda-5 5′,9-837 bp; mda-5 3′, 2531-3365 bp; and GAPDH fragment.

In vitro translation: In vitro translation of mda-5 was performed withNovagen's STP3 kit using T7 RNA polymerase with 35S-Methionine asdescribed in the manufacture's protocol. Template for transcription andtranslation was prepared by BamH1 digestion followed byphenol/chloroform extraction of pGEM-7Zf(+)-mda-5. Proteins that were invitro translated were resolved in 9% SDS-PAGE and detected byautofluorography.

Transient Transfection Assays: 293T cells were plated 1 day prior totransfection and grown to ˜70% confluency. For intracellularlocalization, sterilized cover slips were placed in culture dishes andcells were seeded at 1×105 cells/6 cm tissue culture plate. Transienttransfection assays were performed using SuperFect from Qiagen asdescribed in the manufacturer's protocol. Ten μg of supercoiled plasmidDNA was transfected into 10 cm-tissue culture dish and cells wereharvested two days after transfection.

Western Blot Analysis and Fluorescent Confocal Microscopy: Proteinsamples were prepared from transiently transfected cells by lysis inRIPA buffer supplemented with protease inhibitors. Twenty μg of proteinwas resolved in 9% SDS-PAGE and transferred to nitrocellulose membranes.MDA-5 fusion proteins were probed with either α-HA antibody (BoehringerMannheim) or α-GFP antibody (ClonTech) and HRP-conjugated anti-Mouse IgG(Sigma) and detected by ECL (Amersham). For Fluorescence microscopy,cover glass containing transfected cells were washed with PBS andmounted onto glass slides with mounting medium. Cells were observed withfluorescent confocal microscopy.

Colony-Forming Assays: HO-1 melanoma cells were plated at 8×105 in a6-cm dish one day prior to transfection. Five μg of supercoiledexpression vector DNA was transfected into cells with SuperFect (Qiagen)as described above (18). Two days after transfection, cells wereharvested by trypsinization and replated at 10⁵ cells/6-cm dish withcomplete medium containing 750 μg G418/ml. From each transfection, threedishes were plated. The G418-containing media was replaced once a weekfor three week. Cells were stained with Giemsa and colonies containingmore than 50 cells were counted.

Results

Cloning and Sequence Analysis of mda-5: Subtraction hybridizationbetween a temporally spaced differentiation inducer, IFN-β+mezerein(MEZ), treated HO-1 human melanoma cDNA library and a temporally spaceduntreated control HO-1 cDNA library identified a differentiallyexpressed 0.3 kb EST, melanoma differentiation associated gene-5 (mda-5)((8) Jiang and Fisher, 1993). Northern blotting analysis indicated thatthe mda-5 EST hybridized with a mRNA species of ˜3.8 kb in IFN-β+MEZtreated HO-1 cells ((8) Jiang and Fisher, 1993; Jiang et al., 1994). Afull length mda-5 cDNA containing the complete open reading frame (ORF)was obtained using the C-ORF technique (FIG. 1A) ((16) Kang and Fisher,2000). The ORF of the mda-5 cDNA (3,362 bp excluding the poly A tail)extends from 169 to 3,246 bp and encodes a predicted protein of 1,025amino acids with a molecular mass of 116.7 kDa. Two ATTTA motifs, whichare commonly found in rapid turn-over RNA species, are present atpositions 3,225 and 3,284. A poly A signal (AATAAA) is located 23 bpupstream of the poly A tail. A variant of mda-5, named mda-5p whichcontains an additional 202 bp attached to the 3′ end of mda-5 was alsoidentified by screening a placental cDNA library. Since the poly Asignal for mda-5p is also located 23 bp upstream of its poly A tail,while the ORF remains constant, mda-5p is possibly an alternativelypoly-adenylated variant of mda-5. The existence and tissue specificdistribution of the two variant forms of mda-5 remains to be determined.However, RT-PCR analysis using HO-1 melanoma cells identified only mda-5and not mda-5p.

Electronic sequence analysis of the MDA-5 protein using motif andprofile scans of proteins presently in the protein database identifiedtwo conserved domains, a caspase recruitment domain (CARD) and an RNAhelicase domain. The CARD domain which was defined by generalizedprofile alignment within the RAIDD and ICH-1 amino terminal regions, ispresent in various apoptotic molecules such as Mch6, ICE, ICH-2, c-IAP1,c-IAP2 and Ced-3. Current evidence suggests that the biological role ofCARD is the recruitment of caspase to apoptotic signaling receptorcomplexes (19). The sequence alignment of N-terminal 50 amino acids (aa125-174) of MDA-5 with other CARD-proteins reveals significant sequencehomology at conserved amino acids of CARD (FIG. 1B). MDA-5 displays thehighest homology to the CARD region of RAIDD, which is involved inTNF-R1-mediated apoptotic signal transduction (FIG. 1C) (19). TheC-terminal 100 amino acids (aa 722-823) of MDA-5 also show significantsequence homology to the RNA helicase C-terminal conserved domain, whichis involved in RNA binding and unwinding of double-stranded RNA (FIG.1D) (20). In addition, as with other RNA helicases MDA-5 also containsan ATPase A and B motif (331-TGSGKT and 443-DECH) (FIG. 1D) (20).However, MDA-5 has unique features in its helicase C-terminal motif andATPase A motif. MDA-5 has ARGRA instead of the well-conserved YIHRIGRXXRmotif, which is critical for RNA binding in other RNA helicases (20).The ATPase A motif of MDA-5 (LPTGSGKT) is also different from theconsensus sequence motif (A/GXXGXGKT) found in other RNA helicases (20).Moreover, MDA-5 is the first putative RNA helicase that retains both analtered RNA binding motif and an ATPase A motif. Screening of theSwissProt database for homologous sequences containing both of thesemotifs identified three yeast hypothetical ORFs encoding putativehelicases (Gen Bank Accession Number Q09884, Q58900 and P34529). Theunique features conserved in MDA-5 and these yeast proteins may signifythat MDA-5 is a member of a new family of helicases. RNA helicases areknown to be involved in diverse cellular processes including RNAsplicing, RNA editing,

RNA nuclear cytosolic transport, translation and viral replication byATP-dependent unwinding of dsRNA (20). However, based on the uniquestructure of MDA-5, it is not possible at present to ascribe abiological role for this mew molecule and new family of helicases.

Expression Pattern of mda-5: Since the mda-5 EST was cloned fromdifferentiating HO-1 melanoma cells treated with IFN-β+MEZ, furtherstudies were performed to define the type of molecules capable ofregulating mda-5 expression. For this purpose, HO-1 cells were treatedwith a spectrum of agents affecting growth and differentiation inmelanoma cells, including retinoic acid, mycophenolic acid,12-O-tetradecanoylphobol-13-acetate (TPA) and 3′-5′ cyclic AMP. Theeffect of different types of IFNs and dsRNA (poly IC) and the effect ofgrowth in serum-free medium on mda-5 expression was also evaluated byNorthern blotting analyses. As seen in FIG. 2A, steady-state mda-5message level dramatically increases after treatment with IFN-β ordsRNA. IFN-α (FIG. 4A) and IFN-γ also increase mda-5 transcript levels,but the magnitude of this effect is less than with IFN-β or dsRNA. Sincethe other reagents tested ware not effective inducers of mda-5expression, mda-5 may represent an interferon-responsive, primarilyIFN-β-responsive, gene. Although MEZ treatment by itself does not inducemda-5 expression, it can augment mda-5 expression when used incombination with IFN-β and IFN-γ by approximately 3- to 5-fold,respectively (FIG. 2A). A similar expression pattern of mda-5 as seen inHO-1 cells also occurs in human skin fibroblasts treated with IFN-β,IFN-γ or MEZ alone, or in combination (FIG. 2B). Since MEZ co-treatmentdoes not prolong the half-life of the mda-5 transcript (FIG. 7A),augmentation of IFN-β or IFN-γ-induced mda-5 expression might occur at atranscriptional level, possibly by cross-talk between IFN and MEZsignaling pathways.

The induction of mda-5 expression by IFN-β also occurs in additionalhuman melanoma cells and in normal and tumor cell lines of diverseorigin treated with IFN-5 (FIGS. 3A and 3B). The induction of mda-5expression by IFN-β is independent of the status of p53 and RB. In thiscontext, mda-5 is a bona fide IFN-β-responsive gene that can be inducedin a broad spectrum of normal and tumor cell types irrespective ofgenetic variations present in the different tumor cell lines.

Since IFN signals through membrane receptor associated tyrosine kinases,the inducibility of mda-5 in HO-1 melanoma cells by ligands of othermembrane tyrosine kinase receptors including IL-6, EGF, TGF-α, TGF-β,TNF-α and PDGF was studied by Northern blotting (FIGS. 4A and 4B). Adirect comparison of the potency of induction of mda-5 between differentsub-types of IFN was also evaluated (FIGS. 4A and 4B). IFN-β displayedat least a 10-fold higher potency in mda-5 induction than IFN-α or IFN-γ(FIG. 4A)

2E Among the other ligands of membrane receptors, TNF-α induced mda-5expression at comparable levels as seen with IFN-α (FIG. 4A). A similarpattern of induction of mda-5 expression was also apparent in earlypassage human skin fibroblasts (FIG. 4B). Therefore, induction of mda-5expression by IFN-α, IFN-β, IFN-γ and TNF-α is not unique to HO-1 cells,but rather may represent a general response of this gene in diversecellular contexts. Considering that these agents can produce apoptoticsignals in specific target cells, a possible role for MDA-5 in thisprocess, through its CARD domain, is a possibility.

Treatment of HO-1 cells with IFN-β+MEZ results in terminaldifferentiation and a concomitant irreversible loss in cellularproliferation (Fisher et al., 1985). Terminal differentiation in themajority of inducer-treated cells occurs within 24 hr of treatment. Inthis context, the timing of mda-5 expression can provide a clue to theinvolvement of mda-5 in the induction of differentiation or in themaintenance of the differentiated phenotype. The timing of response totreatment can also provide insights into the mechanism of induction ofmda-5. The timing of mda-5 expression was studied by Northern blottingand mda-5 message level began increasing within 2 hr of treatment withIFN-D or IFN-β+MEZ (FIG. 5). The mda-5 message level peaks between 6-8hr and the elevated level remains elevated over a 96 hr period. AlthoughMEZ further increases mda-5 message level above that observed with IFN-βalone, it does not effect the timing of mda-5 expression. The fastkinetics of mda-5 induction suggested that mda-5 could be an earlyIFN-β-responsive gene and a major component mediating IFN-β inducedgrowth inhibition and antiviral potency. In contrast, MEZ alone orserum-starvation induced lower levels of mda-5 expression and the timingof induction was delayed (first apparent after 48 hr) (FIG. 5). Judgingfrom the delayed kinetics of mda-5 induction by MEZ treatment andserum-deprivation, this induction could be indirect resulting from theproduction of a cellular product(s) during the prolonged duration oftreatment.

Organ-Specificity of mda-5 Expression: The organ-specific expressionpattern of mda-5 was determined by hybridization of this gene with PolyA+ RNA from different organs immobilized on multiple tissue Northernblots (ClonTech)(FIG. 6). Most organs expressed mda-5 at low levelsexcept in the brain and lung in which expression was barely detectable.In testes, a 2.4 kb band instead of a 3.8 kb band present in the otherorgans was detected using the mda-5 probe. However, no organ showednoticeably higher levels of expression of mda-5. The highly induciblenature of mda-5 expression by IFNs, especially IFN-β, and TNF-α indiverse cell types and the relatively low basal message level in variousorgans strongly suggest that mda-5 could play a role in responses thatare specific for IFN signaling, but less critical during normalphysiological processes.

Mechanistic Aspects of mda-5 Induction: Steady state transcript levelsof mda-5 were greatly increased during induction of terminaldifferentiation in HO-1 melanoma cells. The increased mda-5 messagelevel could result from post-transcriptional control, such as messagestabilization, or from enhanced transcription. The time course of decayin IFN-β and IFN-β+MEZ induced mda-5 mRNA levels were determined byblocking transcription with actinomycin D. A gradual temporal decreasein mda-5 transcript level after actinomycin D treatment was observed inboth IFN-β and IFN-β+MEZ treated cells (FIG. 7A). The half-life of mda-5transcript in inducer treated HO-1 cells was approximately 5-6 hr. Sincethe basal level of mda-5 mRNA is too low to monitor quantitatively,effects of IFN-β and IFN-β+MEZ on posttranscriptional control of mda-5message stability could not be determined. However, since actinomycin Dtreatment resulted in a decrease in mda-5 message level the induction ofmda-5 by IFN-β and IFN-β+MEZ could result from changes in the rate oftranscription of this gene. In addition, the fact that the rate of decayin mda-5 message level is not markedly different in IFN-β and IFN-β+MEZtreated cells, mda-5 may also be controlled at a transcriptional levelby MEZ when used in combination with IFN-β. Direct evidence fortranscriptional control of mda-5 expression by IFN-β and IFN-β+MEZtreatment was provided by nuclear run-off assays (FIG. 7B). Treatment ofHO-1 cells with IFN-β greatly increased mda-5 transcription comparedwith only negligible levels of transcription in untreated or MEZ treatedcells. IFN-β+MEZ treatment further enhanced the transcription level ofmda-5-3 fold above that of IFN-β alone. These results document that theincreased steady state levels of mda-5 message that result from IFN-βand IFN-β+MEZ treatment are the primarily the result of increased mda-5transcription. As indicated above, MEZ does not increase transcriptionsignificantly, but MEZ in combination with IFN-β potentiates mda-5transcription. Thus, the ability of MEZ+IFN-β to potentiate mda-5 mRNAlevels most likely results from a synergistic increase in mda-5transcription. Since MEZ is recognized as a weak activator of the enzymeprotein kinase C(PKC), it is possible that a PKC-dependent augmentationof mda-5 transcription that is initiated by IFN-β signaling occursfollowing MEZ treatment.

Specific gene expression changes can be altered in response to asignaling event with or without prior protein synthesis. Certain geneexpression changes (early response genes) including transcriptionfactors and key signaling molecules do not require protein synthesisprior to their expression. By blocking protein synthesis withcycloheximide, a translation inhibitor, it is possible to determinewhether induction of mda-5 expression by appropriate inducer treatmentrequires or is independent of prior protein synthesis (FIG. 7C).Cycloheximide pre-treatment does not inhibit mda-5 steady-state mRNAlevels induced by IFN-α, IFN-β, IFN-γ, TNF-γ and poly IC. Thus, mda-5 isprimary response gene that is regulated by IFN-α, IFN-β, IFN-γ, TNF-γand poly IC treatment. In fact, in certain situations cycloheximidetreatment further increased the level of induction of the mda-5 message.This finding suggests that cycloheximide treatment may inhibit thesynthesis of a protein(s) that destabilizes mda-5 mRNA.

Expression of MDA-5 Protein and Intracellular Localization of MDA-5: Toverify the authenticity of the mda-5 cDNA clone, in vitro translationexperiments were performed. Expression of the mda-5 cDNA in an in vitrotranslation assay results in an encoded protein of ˜120 kDa, close tothe predicted size of the MDA-5 protein which is 116.7 kDa (FIG. 8A).The MDA-5 protein was tagged with either green fluorescent protein (GFP)or hemaglutinnin (HA) and transiently transfected into 293 cells.Western blot analyses of cell lysates specifically detected an ˜120 kDaprotein (HA-tagged) and an ˜160 kDa protein (GFP-tagged) in mda-5 cDNAtransfected cells. These findings indicate that the cloned mda-5 cDNAdoes encode a protein of the expected size for this gene. Confocalfluorescence microscopy of 293T cells transiently transfected withGFP-mda-5 fusion protein demonstrated that the protein localizes in thecytosol (FIG. 8C). A specific localization pattern within the cytoplasmof the GFP-mda-5 fusion protein was not observed. It is conceivable thatthe MDA-5 protein in the cytoplasm may play a role in the translation ofspecific mRNAs.

Effect of mda-5 on Colony Forming Ability of HO-1 Cells: HO-1 cellstreated with IFN-β grow slower and display a noticeable enlargement insize in comparison with untreated cells. Since mda-5 is inducedprimarily by IFN-β, ectopic expression of mda-5 could mimic the effectIFN-β treatment and decrease proliferation. It is also conceivable thatthe CARD region of mda-5 could induce apoptotic signals and that ectopicexpression of mda-5 could affect cell survival. To test for growthinhibitory or pro-apoptotic effects of mda-5 this gene was transfectedand ectopically expressed in HO-1 cells and colony forming ability wasdetermined (FIG. 9). Compared with parental vector transfected cells,the number of G418-resistant colonies in mda-5 expression-vectortransfected cells was reduced by ˜70%. A reduction in colony numbersthat was less dramatic than the full coding frame of mda-5 versusparental vector transfected cells was also apparent when HO-1 cells weretransfected with a deletion mutant of mda-5. Ectopic expression of themda-5 deletion mutant (DN7, D310-484 including both ATPase motifs)caused a 47% reduction and transfection with a 2 kb antisense mda-5(EB11) resulted in a 56% reduction in colony formation versus vectortransfected controls. It appears that antisense mda-5 does noteffectively block mda-5 expression. In fact, endogenous mda-5 expressionwas observed in cells transfected with antisense mda-5. It is possiblethat antisense mda-5 expression induces intracellular dsRNA formationand the dsRNA, in turn, induces endogenous mda-5 expression. In thisway, ectopic expression of antisense mda-5 may affect colony-formingefficiency of HO-1 cells by directly altering the level of mda-5 inthese cells. This is only one hypothetical explanation for thisapparently paradoxical observation. Further studies are necessary todefine the precise mechanism(s) by which ectopic expression of mda-5exerts its effect on colony formatting ability of HO-1 cells.

Mda-5 Promoter Isolation and Characterization: Induction of mda-5 mRNAsubsequent to treatment of human HO-1 melanoma cells with IFN-βindicated the strong likelihood of transcriptional regulation of geneexpression based on Northern blot studies. To determine if the primarylevel of regulation was indeed transcriptional, a nuclear run-onexperiment was performed (FIG. 7). Induction of mda-5 gene expression,as detected by a positive hybridization signal occurred in HO-1 samplesthat had been treated with IFN-β as opposed to a much lower signal inuntreated cells, thereby validating the above hypothesis.

Having confirmed that induction of Mda-5 mRNA occurred primarily at thetranscriptional level, it was decided that regulatory genomic DNAsequences involved in this process should be isolated and characterized.To achieve this goal, a human genomic DNA library constructed in aBacterial Artificial Chromosome vector (BAC, Genome Systems Inc.) wasscreened using the mda-5 cDNA as a probe. Two rounds of screening wereperformed to obtain two overlapping clones that spanned the entire mda-5genomic locus including several thousand bp of sequence upstream of thetranslational initiation codon. Mapping of the BAC clone containing theupstream region by restriction enzyme digestion, Southern blotting andsequence analysis permitted the identification of DNA fragments thatcontained potentially important regulatory sequences, which in the caseof most protein coding genes lie upstream of the transcriptioninitiation site. An approximately 7 kb HindIII fragment containing apartial first exon (including the initiator methionine) andapproximately 6 kb of upstream sequence (FIG. 10) was subcloned into theHindIII site of the promoterless luciferase reporter vector, pGL3(Promega). Transfection of this construct into HO-1 cells in thepresence or absence of IFN-β did not result in the production ofLuciferase enzyme as determined by luminometric quantitation assays,necessitating a re-examination of the cloned DNA sequence. Conceptualtranslation of the cloned sequence when initiated from the mda-5translation initiation ATG site (FIG. 10) indicated that it would causetranslational misreading and premature truncation of the Luciferase openreading frame with subsequent loss of enzymatic activity. To circumventthis problem, a small deletion of DNA sequences containing the mda-5initiator methionine was carried out using a BstXI restriction digestion(FIG. 10) followed by blunt ending the incompatible end overhangs andrecircularization of plasmid by ligation.

The modified mda-5 reporter construct was transfected into parallel setsof HO-1 cells that were treated or not with IFN-β. Quantitation ofluciferase activity indicated that this modified reporter containing apartially deleted Exon 1 and around 6 kb of upstream sequences, showed˜10 fold higher luciferase activity in cells that had been treated withIFN-β compared to untreated controls. This level of induction wascomparable to that seen with the endogenous gene in Northern blotanalyses. It therefore appeared that the cloned regulatory genomic DNAsequence in the reporter construct contained the elements required forthe regulation of the mda-5 gene. It was however, necessary to confirmthat the cloned sequences contained all the regulatory elements involvedin the transcriptional control of the endogenous cellular gene. SinceHO-1 melanoma cells show a very low and variable transfection efficiencyit initially proved very difficult to determine the activity of thetransiently transfected reporter in a consistent way for variables suchas kinetics of induction, optimal concentration of inducer and theidentification of other potential activators. To circumvent thistechnical problem the mda-5 promoter construct was stably integratedinto genomic DNA of HO-1 cells by co-transfection with a puromycinresistance plasmid and selection to isolate a clonal population ofstable integrants. This selection procedure resulted in the productionof several clones of which 48 were randomly picked for further analysis.Screening of these stable promoter clones by treatment with IFN-βindicated that an entire range from completely inactive to highlyactive, as measured by luciferase activity, had been obtained (FIG. 11).Some of the clonal isolates showed induction levels similar to theendogenous gene (around 10 fold), while others displayed much higherinduction (around 100-fold). It is a likely possibility that the clonesshowing higher levels of induction contain multiple copies of integratedplasmid that due to an additive effect show higher levels of activity.Two individual clones (#20 and #40) were selected for further analysesto determine if activation kinetics and overall responsiveness toinducer, as a measure to ascertain the completeness of the isolatedpromoter sequence, mimicked that previously observed for the endogenousgene. In the initial screen (FIG. 11) the clone designated #20 showed avery low basal activity that on induction was >1000 fold. It wastherefore not included in the plot to permit the scale to represent theother clones (ranging from 0-150 fold induction) accurately. This cloneon subsequent analysis displayed a very low basal activity but a muchlesser final fold activation (FIG. 12) than seen in the initial screen,but has maintained this property over several subsequent culturepassages. To determine the induction kinetics of the promoter constructfollowing treatment with IFN-β, a fixed number of cells (106/6 cmculture dish) was treated with inducer and assayed for luciferaseactivity compared with a parallel uninduced control sample, at varioustime-points following treatment (FIG. 12). Irrespective of the finalfold-induction of luciferase levels, which varied in an individualclone, the overall pattern of induction kinetics was almost identicaland similar to that of the endogenous gene as determined by Northernblotting. Similarly, assays were performed to determine the range ofsensitivity of detection of exogenously added Interferon levels asdetermined by a luciferase read out (FIG. 13). The results of this assayclosely paralleled that observed for the endogenous gene with measurablelevels of 0.2 U of IFN-β being detectable. The promoter clone isolateswere also used to determine responsiveness to different forms of IFNsincluding human IFN-α, β, and γ synthetic double stranded RNA(polylC:IC; Amersham) and TNF-α (FIG. 14A) using transient transfectionassays with the reporter construct, in HO-1 melanoma cells. In addition,Clone #40 stable HO-1 cells were treated with human IFN-αA, -αb2, -αC,αD, αF, -αG, αH, -α, -αJ, -αA/D, PBL 1001, Bovine Tau, Ω and Human IFN-β(FIG. 14B). Differential levels of responsiveness were seen dependent onthe type of compound used, in general the Mda-5 promoter construct wasmost responsive to INF-β relative to other IFNs, comparable to theresults obtained in Northern analyses with the endogenous cellular gene.As seen above, Mda-5 gene induction also occurs upon treatment of cellswith synthetic double stranded RNA (poly IC:IC). Studies identical tothose described for IFN-β were performed using double stranded RNA as aninducer with the stable HO-1 promoter clones. These experimentsgenerated results that were similar to endogenous gene induction forparameters including time and level of induction (FIG. 15).

Discussion

Genes displaying differential expression as a function of induction ofterminal differentiation by treatment of HO-1 melanoma cells withIFN-β+MEZ are classified into four subgroups based on their inductionpattern (Jiang and Fisher, 1993). Mda-5 represents a Type I mda gene,which is induced by IFN-β and IFN-β+MEZ. Treatment with MEZ alone, whichis a protein kinase C activator and a weak second-stage tumor promoter,does not induce mda-5 expression, but it potentiates mda-5 expression ata transcriptional level when combined with IFN-β. The inducibleexpression of 21-5′oligoadenylate synthetase, another IFN-responsivegene, by IFN-α is augmented by TPA. Numerous evidence based on the useof PKC inhibitors indicate that the potentiation of IFN-gene expressionby TPA involved activation of PKC, but the exact mechanism of thisinduction remains to be determined.

HO-1 melanoma cells treated with IFN-β increase in size, display slowergrowth kinetics and exhibit enhanced melanogenesis, but they do notundergo obvious morphological changes or cell death (Fisher et al.,1985). In contrast, MEZ, which does not induce mda-5, induces profoundchanges in the morphology of HO-1 cells, including the production ofdendrite-like processes. Reagents that induce specific components ofmelanocytic differentiation in human melanoma cells, including all transretinoic acid (RA), mycophenolic acid (MPA), cyclic-AMP (cAMP), dimethylsulfoxide (DMSO) and TPA, also fail to significantly induce mda-5expression. Therefore, it is possible that the primary role of mda-5 inthe induction of terminal cell differentiation of HO-1 cells byIFN-β+MEZ is restricted to IFN-β-mediated suppression of cellproliferation.

Although IFN-α and IFN-γ significantly induce mda-5 expression, IFN-βwas >5-fold more effective in inducing mda-5 expression than eitherIFN-α or IFN-γ. IFN-β enhanced the expression of mda-5 in normal andtumor cell lines, including various melanoma cell lines regardless oftheir p53 or Rb status. Although mda-5 was undetectable in Northern blotof whole brain Poly A+ mRNA, mda-5 was detected and further induced byIFN-β treatment in cultured normal cerebellum and glioblastomamultiforme cells. In these contexts, mda-5 can be classified as anIFN-β-inducible gene. Both IFN-β and IFN-α share a common receptor(IFN-R1) and often display a similar pattern of gene expression changes,but the biological effects of these agents can be distinct. Mda-5 and agene named INF-R1 are unique in that they display increasedresponsiveness to IFN-β than to IFN-α, which may involve IFN-β-specificcellular processes.

In addition to IFNs, the expression of mda-5 is also induced in bothHO-1 melanoma and human skin fibroblast cells by TNF-α and poly IC. BothTNF-α and poly IC are established inducers of IFN-β gene expression.Based on these facts it is possible that TNF-α and poly IC may inducemda-5 gene expression by modulating IFN-β gene expression. In contrast,pretreatment with cycloheximide (CHX), a protein synthesis inhibitor didnot inhibit mda-5 expression induced by TNF-α or poly IC, which suggeststhat these agents are direct inducers of mda-5. Poly IC directlyactivates PKR (dsRNA-activated interferon-inducible protein kinase) andinduces class I MHC expression. TNF-α signaling was also found to bedependent on PKR activation. Alternatively or additionally, PKR which,independent of IFN receptor signaling, phosphorylates IkB andtransactivates NFkB could be the mediator of TNF-α and poly IC-inducedmda-5 expression. However, it is still possible that TNF-α and poly ICcould stimulate secretion of preexisting IFN-β without the requirementof new protein synthesis.

IFNs were initially identified as molecules that provide immediateprotection against viral infection by eliciting an antiviral state intreated cells. IFN treatment evokes diverse responses depending on thetarget cell which include growth inhibition, changes in differentiation,induction or inhibition of apoptosis and changes in the expression ofimmune system modulating genes. IFN-β displays more potent growthinhibitory effects on normal melanocytes and melanoma cells, includingHO-1, than IFN-α and IFN-γ. Interestingly, the growth inhibitory effectof IFNs in these cells correlates well with the level of induction ofmda-5 expression. In addition, both inducers of mda-5, TNF-α and poly ICinhibit cell proliferation and induce apoptosis in a cell type specificmanner. Induction of mda-5 by IFN-β is an early event, since the steadystate mRNA message levels begin increasing within two hr of treatment.These results suggests that mda-5 may play a pivotal role inIFN-β-mediated suppression of cell proliferation.

Ectopic expression of mda-5 reduces the colony-forming capacity of HO-1melanoma cells by ˜70%. Considering the inefficient nature oftransfection and the random incorporation of transfected genes into thecellular genome, the effect of ectopic mda-5 expression oncolony-forming efficiency is quite dramatic. Surprisingly, theexpression of a deletion mutant of mda-5 (deletion of the ATPase motif)also reduced colony formation (˜47%), but was markedly less potent thanthe wild type mda-5 gene. Colony-forming efficiency is regulated bymultiple parameters including inherent plating efficiency, and thegrowth inhibitory or toxic effect of the transfected gene product.Further studies are required to determine which factor is most criticalin reducing the colony-forming efficiency of cells ectopicallyexpressing mda-5.

Profile scans of the MDA-5 protein reveal putative CARD and RNA helicasemotifs. Multiple sequence alignments of the CARD motif in MDA-5 usingthe ClustalW system indicate that this region most closely resembles theCARD of RAIDD, which is a component of TNF-R1-mediated apoptoticsignaling pathway and which contains both a death domain and a CARDmotif. RAIDD interacts with RIP through its death domain and with ICH-1(caspase-2) probably via its CARD motif. Although not as effective asIFN-β, TNF-α also induces mda-5 expression in HO-1 melanoma cells. In istherefore conceivable that mda-5 may interact with RAIDD and serve as adeath effector molecule like ICH-1. A pro-apoptotic role of mda-5 isalso supported by the dsRNA-dependent induction of this gene, which alsoactivates PKR, a recognized molecule involved in growth suppression andapoptosis. However, a direct apoptotic role of mda-5 does not coincidewith the effect of IFN-β on HO-1 melanoma cells, which results in growthsuppression and not cell death. It is feasible that mda-5 may be acomponent of a death effector molecule, but by itself it lacks thecapacity to trigger apoptosis. In this context, ectopic expression ofmda-S may result in growth inhibition and not apoptosis. It is alsopossible that the level of ectopic expression of mda-5 may determinewhether this molecule is growth inhibitory or toxic. If this is true,expression of mda-5 by means of a adenovirus under the control of astrong promoter may produce sufficiently high levels of MDA-5 to inducecell death as opposed to growth inhibition.

Another distinct motif present in the MDA-5 protein is a RNA helicasesignature domain, which spans the C-terminal half of this molecule. RNAhelicase is a family of enzymes with a helicase motif, which potentiallycatalyzes NTP-dependent dsRNA unwinding activity. Not only are the coreresidues among the RNA helicases conserved, but also the spaces betweenthese residues are retained in the different RNA helicases. Three mainfeatures characterize RNA helicases from the N- to C-terminal, an ATPaseA motif(GXXGXGKT), an ATPase B motif (DEAD, DEAH or DEXH) and a criticaldomain for RNA interaction (HRIGRXXR) (Dong-chul, correct?). RNAhelicases are classified into three subgroups based on their ATPase Bmotifs. RNA helicases are implicated in the majority of steps associatedwith RNA processing and transcription, nuclear and mitochondrial RNAsplicing, RNA editing, ribosomal biogenesis, nuclear cytosolic RNAexport, degradation of nonsense RNA and RNA translation. Hence, RNAhelicases affect many biological phenomena including celldifferentiation, proliferation, development and viral life cycle.Although the RNA helicases are classified into three subgroups, thebiological relevance of these groups remains to be defined. In addition,the enzymatic activity of many putative RNA helicases has not beenconfirmed, this could partly be because of the absence of theappropriate substrate and standard protocol due to the diversity ofthese enzymes.

Despite the well-conserved attributes of RNA helicases, MDA-5 containsfour unique features that could mediate functional divergence. The CARDdomain of MDA-5 in its N-terminal region is not found in any previouslyidentified helicases, although the functional significance of thisregion is currently under investigation. The ATPase A motif of mda-5 isunique and contains LPTGSGKT as opposed to the sequences found in otherRNA helicases (GXXGXGKT) and a mutation of the first glycine residue ofmurine eIF-4A to valine abolishes ATP binding ability. Since leucine isa non-polar amino acid as is valine, but it has a bulkier side chainthan valine, MDA-5 may not bind ATP effectively and, hence, may be anATPase defective helicase or it may require a different energy sourceand/or metals for activity. This property of MDA-5 may explain thereduction in colony forming efficiency by a expression of a mutant ofmda-5 lacking this region of the MDA-5 protein. The HRIGRXXR motif whichis critical for RNA binding in vitro is not well conserved in MDA-5(ARGR1). The functional role of such sequence divergence in the MDA-5protein remains to be determined. Three yeast hypothetical ORFs sharespecific features of MDA-5 including ATPase and RNA binding sites, buttheir biological function has not been ascertained. Complementationassays between these proteins can provide insights on functional andevolutionary relationship among these molecules.

Taken together, the distinctive features of the MDA-5 protein suggestthat this molecule represents a member of a new family of RNA helicases.If this is the case, mda-5 may participate in degradation, translationor inhibition of translation of pro- or anti-apoptotic RNA moleculesthrough its RNA helicase domain. Alternatively, mda-5 might be a signaltransducer between IFN signals and the apoptotic machinery to preparethe cell for viral invasion and dsRNA accumulation. Localization ofGFP-mda-5 fusion protein in the cytoplasm is not contradictory to thishypothesis.

The reporter isolate comprising the mda-5 promoter sequences driving theluciferase cDNA, based on comparison of the quantitation of luciferaseassays to fold induction seen in Northern blot analyses of RNA fromtreated cells, closely mimicked the induction behavior of the endogenousgene. Activation of gene expression occurred primarily with IFN-β anddouble stranded RNA and to a lesser extent with other IFNS. This DNAsequence is therefore of considerable utility in understanding theregulation of mda-5 in particular and IFN-β inducible genes in general,also encompassing but not restricted to the analysis of compoundsincluding synthetic small molecules that affect this pathway.

Due to the high level of sensitivity, technical simplicity andamenability toward semi-automation of luciferase assays, the mda-5promoter clone isolates in HO-1 melanoma cells comprise an additionalvery useful detection and assay system for IFN levels with potentiallysignificant advantages in terms of cost, convenience andreproducibility. Moreover due to the presence of the reporter constructwithin an in vivo biological context, in addition to the ability toquantitate the amount of exogenously added IFNs or determineresponsiveness to specific IFNS, the system is utilizable in the studyof compounds of a diverse nature that potentially impinge on the pathwaywith respect to multiple biological and pharmacological aspects. Theseinclude agonistic or antagonistic effects of a specific compound on theIFN pathway combined with the general biological toxicity of that or acombination of compounds, potentially within the same assay itself. Thepromoter sequence may also be introduced into appropriate cells with anIFN relevant responsiveness similar to that achieved for HO-1 andstudied parallel to those described in the HO-1 human melanoma system beperformed.

In summary, mda-5 is a new IFN-γ inducible putative RNA helicasecontaining a CARD motif. The expression of mda-5 is also induced bygrowth inhibitory and apoptotic signal molecules such as TNF-_and polyIC. Although it was not demonstrated in the present experiments, theability of IFN-β and poly IC to induce mda-5 expression support thepotential for viral induction of this gene. Ectopic expression of mda-5significantly reduces colony-forming efficiency of HO-1 melanoma cellsas expected from the inductive nature and sequence of this gene. Theenzymatic activity of MDA-5 remains to be determined. As mentionedearlier, mda-5 may be a defective RNA helicase and a naturally occurringinhibitor of additional unknown helicases. If this is the case, it willbe important to identify counterparts of mda-5 which display antiviral,proliferation inhibitory and/or apoptotic roles in cellular physiology.Of particular note is that viruses like hepatitis C virus (HCV) containa helicase in their genome. Defining the enzymatic activity of MDA-5 maybe achieved by modulating the experimental conditions, i.e., by changingreaction conditions including NTP and metal requirements, usingpotential stimulators like 2′-5′ oligoadenylate, etc. Investigation ofthe physiological role and molecular basis of mda-5 action shouldprovide important insights into the mechanism of cellular defenseconferred by IFN against viral attack. This information should provevaluable in developing new strategies for inhibiting viral pathogenicityand for designing more effective antiviral therapeutics.

Example 2 Reporter Cell Lines

Reporter cell lines derived from the HO-1 human melanoma cell linecontaining genomically integrated copies of te Melanoma DifferentiaionAssociated Gene-5 (Mda-5) upstream promoter sequences.

A Bacterial Artificial Chromosome (BAC), human genomic library wasscreened to isolate sequenes containing the Mda-5 gene. Two BAC clonescontaining the coding and upstream sequences of te gene were isolatedand characterized.

The complete intron/exon structure of the coding sequences has beendetermined. An approximately 6 kb fragment upstream of the transcriptionstart site was also isolated. T is fragment was cloned into apromoterless luciferase vector (pGL3 Basic, Promega) and assayed bytransient transfection assays for transcriptional activity. The activitydisplayed by this promoter construct was identical to that of theendogenous gene in terms of responsiveness to inducers (recombinanthuman β-interferon or synthetic double stranded RNA, poly IC) and timekinetics of induction.

Several sublines containing stably integrated copies of thetranscriptionally active luciferase plasmid in a HO-1 human melanomabackground was constructed. Independent clonally isolated colonies wereexpanded and assayed for luciferase activity in the presence ofrecombinant human β-interferon or synthetic double stranded RNA, polyIC. These clones exhibited luciferase activity similar to the endogenousgene except that the level of induction varied from 10 to 100 fold,probably dependent on the number of integrated copies for each clone(the endogenous gene is induced about 10 fold). While these clones aremost responsive to recombinant human β-interferon or synthetic doublestranded RNA, poly IC they are also induced at lower levels by otherinterferons.

These cell lines may be used for:

A. Quantitation of biologically active amounts of interferon produced byvarious procedures;

B. In rapid high throughput screens to determine or distinguish therelative efficacy of compounds agonistic or antagonistic to theinterferon biochemical and signalling pathway;

C. In a rapid high throughput screen to detect small molecules ofpotential pharmacological and therapeutic utility that synergizes orboosts cellular interferon pathways.

Example 3 Full Length Human Mda-5 Promoter

       HindIII              |TCCACTCAATATAAAGCTTGCACTCATTCTCCAAGCCCAGGTGTGATCCGATTCTTCCAG 1---------+---------+---------+---------+---------+---------+ 60AGGTGAGTTATATTTCGAACGTGAGTAAGAGGTTCGGGTCCACACTAGGCTAAGAAGGTCTATACCAAGTCAAGAACGTGGGATACAGAAAGCCCTCTGTCCTTGAGACAATGTAGAGGG 61---------+---------+---------+---------+---------+---------+ 120ATATGGTTCAGTTCTTGGACCCTATGTCTTTCGGGAGACAGGAACTCTGTTACATCTCCCTCTAACTGAGCTTGTTAACACAAGCCACCTATAGACAGCAAAACTAAAAGATCACCCTGT 121---------+---------+---------+---------+---------+---------+ 180AGATTGACTCGAACAATTGTGTTCGGTGGATATCTGTCGTTTTGATTTTCTAGTGGGACAAACACACGCCCACTGAGGCTTCAGAAGCTGTAAACATCCACCCCTAGACACTGCCGTGGG 181---------+---------+---------+---------+---------+---------+ 240TTGTGTGCGGGTGACTCCGAAGTCTTCGACATTTGTAGGTGGGGATCTGTGACGGCACCCTCGGAGCCCCACAGCCTGCCCATCTGCAGGCTCCCCTAGAGGTTTGAGCAGTGGGGCACT 241---------+---------+---------+---------+---------+---------+ 300AGCCTCGGGGTGTCGGACGGGTAGACGTCCGAGGGGATCTCCAAACTCGTCACCCCGTGAGAAGAAGCGAGCCACACCCCCATACTGCCCAAGGTAATTTACAGATTCAATGCCATCCCC 301---------+---------+---------+---------+---------+---------+ 360CTTCTTCGCTCGGTGTGGGGGTATGACGGGTTCCATTAAATGTCTAAGTTACGGTAGGGGATCAAGCTACCAATGACTTTCTTCACAGAATTGGAAAAAACTACTTTAAAGTTCATATGG 361---------+---------+---------+---------+---------+---------+ 420TAGTTCGATGGTTACTGAAAGAAGTGTCTTAACCTTTTTTGATGAAATTTCAAGTATACCAACCAAAAAAGAGCCCGCATCGCCAAGTCAATCCTAAGCCAAAAGAACAAAGCTGGAGGC 421---------+---------+---------+---------+---------+---------+ 480TTGGTTTTTTCTCGGGCGTAGCGGTTCAGTTAGGATTCGGTTTTCTTGTTTCGACCTCCGATCACCCTACCTGACTTCAAACAATACTACAAGGCTACAGTAACCAAAACAGCATGGTAC 481---------+---------+---------+---------+---------+---------+ 540TAGTGGGATGGACTGAAGTTTGTTATGATGTTCCGATGTCATTGGTTTTGTCGTACCATGTGGTACCAAAACAGAGATATAGATCAATGGAACAGAACAGAGCCCTCAGAAATAATGCCA 541---------+---------+---------+---------+---------+---------+ 600ACCATGGTTTTGTCTCTATATCTAGTTACCTTGTCTTGTCTCGGGAGTCTTTATTACGGTCATATCTACAACTATCTGATCTTTGACAAACCTGAGAAAAACAAGCAATGGGGAAAGTAT 601---------+---------+---------+---------+---------+---------+ 660GTATAGATGTTGATAGACTAGAAACTGTTTGGACTCTTTTTGTTCGTTACCCCTTTCATATCCCTATTTAATAAATGGTGCTGGGAAAACTGGCTAGCCATATGTAGAAAGCTGAAACTG 661---------+---------+---------+---------+---------+---------+ 720AGGGATAAATTATTTACCACGACCCTTTTGACCGATCGGTATACATCTTTCGACTTTGACGGTTCCTTCCTTACACCTTATACAAAAATCAATTCAAGATGGATTAAAGACTTAAACGTT 721---------+---------+---------+---------+---------+---------+ 780CCAAGGAAGGAATGTGGAATATGTTTTTAGTTAAGTTCTACCTAATTTCTGAATTTGCAAAGACCTAAAACCATAAAAACCCTAGAAGAAAACCTAGGCATTACCATTCAGGACATACGC 781---------+---------+---------+---------+---------+---------+ 840TCTGGATTTTGGTATTTTTGGGATCTTCTTTTGGATCCGTAATGGTAAGTCCTGTATGCGATGGGCAAGGACTTCATGTCTAAAACACCAAAAGCAATGGCAACAAAAGCCAAAATTGAC 841---------+---------+---------+---------+---------+---------+ 900TACCCGTTCCTGAAGTACAGATTTTGTGGTTTTCGTTACCGTTGTTTTCGGTTTTAACTGAAACGGTATCTAATTAAACTAAAGAGCTTCTGCACAGCAAAAGAAACTACCATTAGAGTG 901---------+---------+---------+---------+---------+---------+ 960TTTGCCATAGATTAATTTGATTTCTCGAAGACGTGTCGTTTTCTTTGATGGTAATCTCACAACAGGCAACCTACAAAATGGGAGAAAATTTTCGCAACCTACTCATCCGACAAAGGGCTA 961---------+---------+---------+---------+---------+---------+ 1020TTGTCCGTTGGATGTTTTACCCTCTTTTAAAAGCGTTGGATGAGTAGGCTGTTTCCCGATATATCCAGAATCTACAATGAACTCAAACAAATTTACAAGAAAAAAACAAACAACCCCATC 1021---------+---------+---------+---------+---------+---------+ 1080TATAGGTCTTAGATGTTACTTGAGTTTGTTTAAATGTTCTTTTTTTGTTTGTTGGGGTAGAAAAAGTGGGTGAAGGACATGAACAGACACTTGTCAAAAGAAGACATTTATGCAGCCAAA 1081---------+---------+---------+---------+---------+---------+ 1140TTTTTCACCCACTTCCTGTACTTGTCTGTGAACAGTTTTCTTCTGTAAATACGTCGGTTTAAACACATGAAAAAATGCTCACCATCACTGGCCATCAGAGAAATGCAAATCAAAACCACA 1141---------+---------+---------+---------+---------+---------+ 1200TTTGTGTACTTTTTTACGAGTGGTAGTGACCGGTAGTCTCTTTACGTTTAGTTTTGGTGTATGAGATACCATCTCACACCAGTTAGAATGGCAATCATTAAAAAGTCAGGAAACAACAGG 1201---------+---------+---------+---------+---------+---------+ 1260TACTCTATGGTAGAGTGTGGTCAATCTTACCGTTAGTAATTTTTCAGTCCTTTGTTGTCCTGATGGAGAGGATGTGGAGAAATAGGAACACTTTTGCACTGTTGGTGGGACTGTAAACTA 1261---------+---------+---------+---------+---------+---------+ 1320ACTACCTCTCCTACACCTCTTTATCCTTGTGAAAACGTGACAACCACCCTGACATTTGATGTTCAACCATTGTGGAAGTCAGTGTGGTGATTCCTCAGGGATCTAGAACTAGAAATACCA 1321---------+---------+---------+---------+---------+---------+ 1380CAAGTTGGTAACACCTTCAGTCACACCACTAAGGAGTCCCTAGATCTTGATCTTTATGGTTTTGACCCAGCCATCCCATTACTGGGTATATACTCAAAGGACTATAAATCTTGCTGCTAT 1381---------+---------+---------+---------+---------+---------+ 1440AAACTGGGTCGGTAGGGTAATGACCCATATATGAGTTTCCTGATATTTAGAACGACGATAAAAGACACATGCACATGTATGTTTATTGTGGCATTATTCACAATAGCAAAGACTTGGAAC 1441---------+---------+---------+---------+---------+---------+ 1500TTTCTGTGTACGTGTACATACAAATAACACCGTAATAAGTGTTATCGTTTCTGAACCTTGCAACCCAAATGTCCAACAGTGATAGACTGGATTAAGAAAATGTGGCACACATACACCATG 1501---------+---------+---------+---------+---------+---------+ 1560GTTGGGTTTACAGGTTGTCACTATCTGACCTAATTCTTTTACACCGTGTGTATGTGGTACGAATACTATGCAGCCATAAAAAATGATGAGTTCATGTCCTTTGTAGGGACATGGATGAAA 1561---------+---------+---------+---------+---------+---------+ 1620CTTATGATACGTCGGTATTTTTTACTACTCAAGTACAGGAAACATCCCTGTACCTACTTTTTGGAAATCATCATTCTCAGTAAACTATCGCAAGAACAAAAAACCAAACACCGCATATTC 1621---------+---------+---------+---------+---------+---------+ 1680AACCTTTAGTAGTAAGAGTCATTTGATAGCGTTCTTGTTTTTTGGTTTGTGGCGTATAAGTCACTCATAGGTGGGAATTGAACAATGCGAACACATGGACACAGGAAGGAGAACATCACA 1681---------+---------+---------+---------+---------+---------+ 1740AGTGAGTATCCACCCTTAACTTGTTACGCTTGTGTACCTGTGTCCTTCCTCTTGTAGTGTCTCTGGGGACTGTTGTGGGGTGGGGGGAGGGGGGAGGGATAGCATTGGTAGATATACCTA 1741---------+---------+---------+---------+---------+---------+ 1800GAGACCCCTGACAACACCCCACCCCCCTCCCCCCTCCCTATCGTAACCATCTATATGGATATGCTAGATGACGAGTTAGTGGGTGCAGCGCACCAGCATGACACATGTATACATATGTAA 1801---------+---------+---------+---------+---------+---------+ 1860TACGATCTACTGCTCAATCACCCACGTCGCGTGGTCGTACTGTGTACATATGTATACATTCCAACCTGCACATTGTGCACATGTACCCTAAAACTTAAAGTATAATAATAAATAAATAAA 1861---------+---------+---------+---------+---------+---------+ 1920GGTTGGACGTGTAACACGTGTACATGGGATTTTGAATTTCATATTATTATTTATTTATTTTAAATAAATAAATAAATAAAGTAAAATAAAACAATTACAATCTAGCCTTTGAGGTAAAAG 1921---------+---------+---------+---------+---------+---------+ 1980ATTTATTTATTTATTTATTTCATTTTATTTTGTTAATGTTAGATCGGAAACTCCATTTTCTACTGTTTTTCACAAAAACATTTGCAGGTAACTGTTTTTGAAAAGACTTTAAGCTATGGA 1981---------+---------+---------+---------+---------+---------+ 2040ATGACAAAAAGTGTTTTTGTAAACGTCCATTGACAAAAACTTTTCTGAAATTCGATACCTAGGAGTACTTGAAAAATGAATGTTCCAAAACTTATCTATTGATACGTGACTTTCATTTTT 2041---------+---------+---------+---------+---------+---------+ 2100TCCTCATGAACTTTTTACTTACAAGGTTTTGAATAGATAACTATGCACTGAAAGTAAAAATGCCAAAACTGCTATGTAGAAAAGTTTTTATATGTGAAAACTTAAAAACCAGAATTTTAA 2101---------+---------+---------+---------+---------+---------+ 2160ACGGTTTTGACGATACATCTTTTCAAAAATATACACTTTTGAATTTTTGGTCTTAAAATTTTGAATTGGTGAAAGTGATTAGGAAATTATTATCAAGATTTAGTGAACTTAGCCATAATT 2161---------+---------+---------+---------+---------+---------+ 2220AACTTAACCACTTTCACTAATCCTTTAATAATAGTTCTAAATCACTTGAATCGGTATTAATTTTTTCTATTTTAGGCTTACTACTATTTTTGAAATAAAAAGCTACGACAGTATCCTTTT 2221---------+---------+---------+---------+---------+---------+ 2280AAAAAAGATAAAATCCGAATGATGATAAAAACTTTATTTTTCGATGCTGTCATAGGAAAAAATAAACTTTCCTGCTAAATCAGCCTATCAGTTTCAGTTAAATGGCTGAAAGTCTTGCTT 2281---------+---------+---------+---------+---------+---------+ 2340TTATTTGAAAGGACGATTTAGTCGGATAGTCAAAGTCAATTTACCGACTTTCAGAACGAAAAAGTCTCAGTTAAATGGCTAGCTATTATATAGTGTTTATATGTATGTGTGTATATATAT 2341---------+---------+---------+---------+---------+---------+ 2400TTTCAGAGTCAATTTACCGATCGATAATATATCACAAATATACATACACACATATATATAATATATATATATATATATATATATATATATATATATATATATGTAACTAAATTTTTCCTT 2401---------+---------+---------+---------+---------+---------+ 2460TATATATATATATATATATATATATATATATATATATATATACATTGATTTAAAAAGGAATATAAATTGTGCATTCTTTGAAGACTAGCACCGCACCATCTCTTCTTTAATTTTTATATA 2461---------+---------+---------+---------+---------+---------+ 2520ATATTTAACACGTAAGAAACTTCTGATCGTGGCGTGGTAGAGAAGAAATTAAAAATATATAGCGTAGTGGGCTGGAGTCACATATTGGGCACATAAACATGCCAGGCTGGTGCTAGTGTG 2521---------+---------+---------+---------+---------+---------+ 2580TCGCATCACCCGACCTCAGTGTATAACCCGTGTATTTGTACCGTCCGACCACGATCACACTTACAGTCTATCCTTAGAACAAACTTCTGACATGATACCAGAATCTTTCCATTTTACAAC 2581---------+---------+---------+---------+---------+---------+ 2640AATGTCAGATAGGAATCTTGTTTGAAGACTGTACTATGGTCTTAGAAAGGTAAAATGTTGTGATGTATTTGAGGTGATTTTTCAAAGCACAGCAATTAAGAAATAGTATTGAGATGTGAA 2641---------+---------+---------+---------+---------+---------+ 2700ACTACATAAACTCCACTAAAAAGTTTCGTGTCGTTAATTCTTTATCATAACTCTACACTTCTCAGACAGCCTGAACTCAGAGTCTCTGTGCTTAACCATACCCCACACTGCCAGGTTAAG 2701---------+---------+---------+---------+---------+---------+ 2760GAGTCTGTCGGACTTGAGTCTCAGAGACACGAATTGGTATGGGGTGTGACGGTCCAATTCAGCATCTAACACTTTAAATTACACAAAGCAGGCTCATTATTGATACAAATGAGCAAACAA 2761---------+---------+---------+---------+---------+---------+ 2820TCGTAGATTGTGAAATTTAATGTGTTTCGTCCGAGTAATAACTATGTTTACTCGTTTGTTGTAAAGGAACAGAACAACAATTCCAGGGTTTCTCACTAAACTAAAATTATTGTCATTTTC 2821---------+---------+---------+---------+---------+---------+ 2880CATTTCCTTGTCTTGTTGTTAAGGTCCCAAAGAGTGATTTGATTTTAATAACAGTAAAAGTTTGAAAAAGACATTATTGCTATGCATGGTCGTTAAATTGTAGTGGCAGCTCATATTGTT 2881---------+---------+---------+---------+---------+---------+ 2940AAACTTTTTCTGTAATAACGATACGTACCAGCAATTTAACATCACCGTCGAGTATAACAAACTACTTCTTAAAAACTCAAATGAAAAGTTGCATAACAATGGGAAAATACATAGTTCAGC 2941---------+---------+---------+---------+---------+---------+ 3000TGATGAAGAATTTTTGAGTTTACTTTTCAACGTATTGTTACCCTTTTATGTATCAAGTCGAGGATCTCCTGCCTCAAAAGAGAAAGGAAAAAGAAACTTACATTTGGGAACTGGTGAAAA 3001---------+---------+---------+---------+---------+---------+ 3060TCCTAGAGGACGGAGTTTTCTCTTTCCTTTTTCTTTGAATGTAAACCCTTGACCACTTTTGGATTAAAATGAAACCTAGTAGAAGAAACTTGACAGAGGAAAACAATTAATTACTCAAGT 3061---------+---------+---------+---------+---------+---------+ 3120CCTAATTTTACTTTGGATCATCTTCTTTGAACTGTCTCCTTTTGTTAATTAATGAGTTCAGAAAAACAGAAAATAAACTAAATCATGATGCAAAAAATATAGATGAAAAAAGGATACATT 3121---------+---------+---------+---------+---------+---------+ 3180CTTTTTGTCTTTTATTTGATTTAGTACTACGTTTTTTATATCTACTTTTTTCCTATGTAAGTGAGAGATTGTGTCTTGGCTTTTGTTTCCTTAACCTCCTTTCTCCAAAAAGGGTCCCAT 3181---------+---------+---------+---------+---------+---------+ 3240CACTCTCTAACACAGAACCGAAAACAAAGGAATTGGAGGAAAGAGGTTTTTCCCAGGGTACAAGACTATGGGAGATTCCTAAAAAAGAAGTCCCTTCCACCCACACCTAATCCTCATCAC 3241---------+---------+---------+---------+---------+---------+ 3300GTTCTGATACCCTCTAAGGATTTTTTCTTCAGGGAAGGTGGGTGTGGATTAGGAGTAGTGTCAGACCTCATCCAGCAGAGAGACTCCTACTTGTGAGAAAATATGAATTGTTATTGTTGG 3301---------+---------+---------+---------+---------+---------+ 3360AGTCTGGAGTAGGTCGTCTCTCTGAGGATGAACACTCTTTTATACTTAACAATAACAACCGTATTATGTGATGCTAATAGGGTTAGAGGAGGATGACTATTTGGGAAATCAACCTGTGAA 3361---------+---------+---------+---------+---------+---------+ 3420CATAATACACTACGATTATCCCAATCTCCTCCTACTGATAAACCCTTTAGTTGGACACTTACTGTAATATACATTATTATGTAGATTTACTATGGTCTTCAGGGCATTTATCCTCACCTG 3421---------+---------+---------+---------+---------+---------+ 3480TGACATTATATGTAATAATACATCTAAATGATACCAGAAGTCCCGTAAATAGGAGTGGACCACATTGCATATTTTTTAGTCATTACTTACCATCTATCTTCCCACTCCCATTAGAATGTG 3481---------+---------+---------+---------+---------+---------+ 3540GTGTAACGTATAAAAAATCAGTAATGAATGGTAGATAGAAGGGTGAGGGTAATCTTACACAACTCCATAAAAGTAGGAGCTTTGTTAATTTTATTAACTGCACCTAGATCAGTGCTGGTC 3541---------+---------+---------+---------+---------+---------+ 3600TTGAGGTATTTTCATCCTCGAAACAATTAAAATAATTGACGTGGATCTAGTCACGACCAGATGTAATAGATACTTAATAAACATATTTTAAATGACTAGATGATACAATGAATGATATAA 3601---------+---------+---------+---------+---------+---------+ 3660TACATTATCTATGAATTATTTGTATAAAATTTACTGATCTACTATGTTACTTACTATATTTTTGAATGCCAAATATTTAAATATCTTTGGTTTAAATGTTTATTATTTGAGAACAGGTCA 3661---------+---------+---------+---------+---------+---------+ 3720AAACTTACGGTTTATAAATTTATAGAAACCAAATTTACAAATAATAAACTCTTGTCCAGTAATACCAAACATTTGATCCTTTCTCTTCCAGAGCAACAATTAAGTGGTATGAAGAAAATA 3721---------+---------+---------+---------+---------+---------+ 3780TTATGGTTTGTAAACTAGGAAAGAGAAGGTCTCGTTGTTAATTCACCATACTTCTTTTATACATTAACTGGTTCCCCATATTCAGTCAGCAACCCCTTCTCATTCCCCCATGTTTGAAAC 3781---------+---------+---------+---------+---------+---------+ 3840TGTAATTGACCAAGGGGTATAAGTCAGTCGTTGGGGAAGAGTAAGGGGGTACAAACTTTGCAAGAAACAGAAGGATAAGTGCCAGGAAAAAGAATGTTTTTTGGTTTGTTAGTTTGTGCT 3841---------+---------+---------+---------+---------+---------+ 3900GTTCTTTGTCTTCCTATTCACGGTCCTTTTTCTTACAAAAAACCAAACAATCAAACACGATTAACATGTTGAATAAAACCCACTGGCAGCTGGGGGATAGGAGTATGTTTTTGCAACAGC 3901---------+---------+---------+---------+---------+---------+ 3960AATTGTACAACTTATTTTGGGTGACCGTCGACCCCCTATCCTCATACAAAAACGTTGTCGCTTAAAAGATATTTTCATAGACCCAATACTTAAAATTAATAATTTGAGTGCTTTTGTAGA 3961---------+---------+---------+---------+---------+---------+ 4020GAATTTCTATAAAAGTATCTGGGTTATGAATTTTTAATTATTAAACTCACGAAAACATCTAACATCTAATTGGATTATCCTCTATCTGGGTACAAGGTCATCTCCCAAAATTAAGTGAAA 4021---------+---------+---------+---------+---------+---------+ 4080TTGTAGATTAACCTAATAGGAGATAGACCCATGTTCCAGTAGAGGGTTTTAATTCACTTTAAGGAGTAGGGTTCTGTGAAGAAACAGAAAAGAACAGTATAAATCAGGCCTACCTGCAAG 4081---------+---------+---------+---------+---------+---------+ 4140TTCCTCATCCCAAGACACTTCTTTGTCTTTTCTTGTCATATTTAGTCCGGATGGACGTTCCCCAAGGTTTCATTTACTTCAACTTTCAGTGTATTTAACATTATGCCAGCTGCTATGGTC 4141---------+---------+---------+---------+---------+---------+ 4200GGGTTCCAAAGTAAATGAAGTTGAAAGTCACATAAATTGTAATACGGTCGACGATACCAGAACTCAAATACAACTCCCAGGAGAGATGTCATCAAGAGCCCACCAGTTGTGAGTAGTGTA 4201---------+---------+---------+---------+---------+---------+ 4260TTGAGTTTATGTTGAGGGTCCTCTCTACAGTAGTTCTCGGGTGGTCAACACTCATCACATCTAGTTACTATGTAAATATATCCCTTCTTCAAGCAGCTTACAATCCTTCAGGGGTAGAAA 4261---------+---------+---------+---------+---------+---------+ 4320GATCAATGATACATTTATATAGGGAAGAAGTTCGTCGAATGTTAGGAAGTCCCCATCTTTAAGCCTTGCTACATAAGATAATTAGAGAATAAAATAAGACATGTTACCATAAAGTGCTCA 4321---------+---------+---------+---------+---------+---------+ 4380TTCGGAACGATGTATTCTATTAATCTCTTATTTTATTCTGTACAATGGTATTTCACGAGTTTTGGATATTTTGTATGCCCCTAGTAAACACTCACCAAGACTCTGTACTTCTATTATCCT 4381---------+---------+---------+---------+---------+---------+ 4440AAACCTATAAAACATACGGGGATCATTTGTGAGTGGTTCTGAGACATGAAGATAATAGGAGTTCAAAGCACTATCAGGTTTTCCTGGCCTACACAGACTTTATTGAATGTACTTTGCTAA 4441---------+---------+---------+---------+---------+---------+ 4500CAAGTTTCGTGATAGTCCAAAAGGACCGGATGTGTCTGAAATAACTTACATGAAACGATTCAGATTATTTTTCCTAAATATGTCTCTTTGATAACCTAAATGATCTCTCCATCCTTTATA 4501---------+---------+---------+---------+---------+---------+ 4560GTCTAATAAAAAGGATTTATACAGAGAAACTATTGGATTTACTAGAGAGGTAGGAAATATTAATTCTGGACCATGAGATTCTQGTTATGGTGCGTATGTGCCTACCACCCACAGTCACAT 4561---------+---------+---------+---------+---------+---------+ 4620ATTAAGACCTGGTACTCTAAGATCAATACCACGCATACACGGATGGTGGGTGTCAGTGTAGTGGCTACAGAATGCCTTCAGAATGAGTAGTAACCTTAAGGACTCACATTTATGTGGCTT 4621---------+---------+---------+---------+---------+---------+ 4680CACCGATGTCTTACGGAAGTCTTACTCATCATTGGAATTCCTGAGTGTAAATACACCGAACTGTACCAAAATGAAGCTGCCATTTTTCAGTGTGAATATGTTTTTTTTCTCTCATGACAT 4681---------+---------+---------+---------+---------+---------+ 4740GACATGGTTTTACTTCGACGGTAAAAAGTCACACTTATACAAAAAAAAGAGAGTACTGTAAGACAAATGTTGATGTTTACTACAAGTTGGTACATTAGTTGCTAATTAAGTTCCTAGCTG 4741---------+---------+---------+---------+---------+---------+ 4800TCTGTTTACAACTACAAATGATGTTCAACCATGTAATCAACGATTAATTCAAGGATCGACCTCCAGCCAAAACTTGCTGTATTGAATCCAAGAAAAGAATGGCAGCTATATCAAAAATAA 4801---------+---------+---------+---------+---------+---------+ 4860GAGGTCGGTTTTGAACGACATAACTTAGGTTCTTTTCTTACCGTCGATATAGTTTTTATTGTTGTTGGGGGATTTTTTTGTTTTGTTTTATTAAAGGAAAGTTGTATATTAAAGAATATA 4861---------+---------+---------+---------+---------+---------+ 4920CAACAACCCCCTAAAAAAACAAAACAAAATAATTTCCTTTCAACATATAATTTCTTATATGGGAACTTACAAGCTGGGATCTAGGAAACTTTAAGTCTTGGCTTCCTTCTAAGCTGAGTT 4921---------+---------+---------+---------+---------+---------+ 4980CCCTTGAATGTTCGACCCTAGATCCTTTGAAATTCAGAACCGAAGGAAGATTCGACTCAAGGTGGTTCAAGTCCATCCACATCTGTTACCAGGTCCTGGTCAAAGCTGCATAAATACCAG 4981---------+---------+---------+---------+---------+---------+ 5040CCACCAAGTTCAGGTAGGTGTAGACAATGGTCCAGGACCAGTTTCGACGTATTTATGGTCCAATCTAAATATGAGGCAGTAAAGTTAACTGTTTATTGTTACTCACTTTTTCGAACCCAC 5041---------+---------+---------+---------+---------+---------+ 5100GTTAGATTTATACTCCGTCATTTCAATTGACAAATAACAATGAGTGAAAAAGCTTGGGTGCTCCAAATTCCCAGGGAAACAAGTTAGTGTTTGGGAACCCACAGGAGGTCAGGTTTATTT 5101---------+---------+---------+---------+---------+---------+ 5160GAGGTTTAAGGGTCCCTTTGTTCAATCACAAACCCTTGGGTGTCCTCCAGTCCAAATAAATAGGAAGGACTTCCTCCTGTCTTCTCCACATCTCTGCAAAGATGTCTTGTGAGCTTCATC 5161---------+---------+---------+---------+---------+---------+ 5220ATCCTTCCTGAAGGAGGACAGAAGAGGTGTAGAGACGTTTCTACAGAAGACTCGAAGTAGTCTCACCTGTCCCTCGCAGTCTCACCACCCTCAGCCAGGCCTGCCTACATTCACCAGCCG 5221---------+---------+---------+---------+---------+---------+ 5280AGAGTGGACAGGGAGCGTCAGAGTGGTGGGAGTCGGTCCGGACGGATGTAAGTGGTCGGCAGGGTAACTCCCTGTTCACGTCCGGGTCTGTGGCAGTTTCTGTTCACTTCCCCTTTGGAA 5281---------+---------+---------+---------+---------+---------+ 5340TCCCATTGAGGGACAAGTGCAGGCCCAGACACCGTCAAAGACAAGTGAAGGGGAAACCTTAGTCCCAAATCACATGCTTTTATGCCCTGCACATTTTGGCCTACAAAGGACCTTATTGTT 5341---------+---------+---------+---------+---------+---------+ 5400TCAGGGTTTAGTGTACGAAAATACGGGACGTGTAAAACCGGATGTTTCCTGGAATAACAAAAGGCAGAACCTGCTGGGAAAACAAAATATCCGCCGGAGGAGCTTTGCTAGAGCGTTGGT 5401---------+---------+---------+---------+---------+---------+ 5460TTCCGTCTTGGACGACCCTTTTGTTTTATAGGCGGCCTCCTCGAAACGATCTCGCAACCA          EcoRI               |CTTGGTGTCAGAGAGAATTCGCTTTCCTTTTCTGTTTCCCGCGGTGTCCTTAACCAAAGG 5461---------+---------+---------+---------+---------+---------+ 5520GAACCACAGTCTCTCTTAAGCGAAAGGAAAAGACAAAGGGCGCCACAGGAATTGGTTTCCCCTCCTCTCTTCACCCGCCCCGACCAAAAGGTGGCGTCTCCCTGAGGAAACTCCCTCCCC 5521---------+---------+---------+---------+---------+---------+ 5580GGAGGAGAGAAGTGGGCGGGGCTGGTTTTCCACCGCAGAGGGACTCCTTTGAGGGAGGGGGCCAGGCAGATTACGTTTACAAAGTCCTGAGAAGAGAATCGAAACAGAAACCAAAGTCAG 5581---------+---------+---------+---------+---------+---------+ 5640CGGTCCGTCTAATGCAAATGTTTCAGGACTCTTCTCTTAGCTTTGTCTTTGGTTTCAGTCGCAAACTCTGTAAGAACTGCCTGACAGAAAGCTGGACTCAAAGCTCCTACCCGAGTGTGC 5641---------+---------+---------+---------+---------+---------+ 5700CGTTTGAGACATTCTTGACGGACTGTCTTTCGACCTGAGTTTCGAGGATGGGCTCACACGAGCAGGATCGCCCCGGTCCGGGACCCCAGGCGCACACCGCAGAGTCCAAAGTGCCGCGCC 5701---------+---------+---------+---------+---------+---------+ 5760TCGTCCTAGCGGGGCCAGGCCCTGGGGTCCGCGTGTGGCGTCTCAGGTTTCACGGCGCGGTGCCGGCCGCACCTGCCTGCCGCGGCCCCGCGCGCCGCCCCGCTGCCCACCTGCCCGCCT 5761---------+---------+---------+---------+---------+---------+ 5820ACGGCCGGCGTGGACGGACGGCGCCGGGGCGCGCGGCGGGGCGACGGGTGGACGGGCGGAGCCCACCTGCCCAGGTGCGAGTGCAGCCCCGCGCGCCGGCCTGAGAGCCCTGTGGACAAC 5821---------+---------+---------+---------+---------+---------+ 5880CGGGTGGACGGGTCCACGCTCACGTCGGGGCGCGCGGCCGGACTCTCGGGACACCTGTTGCTCGTCATTGTCAGGCACAGAGCGGTAGACCCTGCTTCTCTAAGTGGGCAGCGGACAGCG 5881---------+---------+---------+---------+---------+---------+ 5940GAGCAGTAACAGTCCGTGTCTCGCCATCTGGGACGAAGAGATTCACCCGTCGCCTGTCGC                                      BstXI                                          |GCACGCACATTTCACCTGTCCCGCAGACAACAGCACCATCTGCTTGGGAGAACCCTCTCC 5941---------+---------+---------+---------+---------+---------+ 6000CGTCCGTGTAAAGTGGACAGGGCGTCTGTTGTCGTGGTAGACGAACCCTCTTGGGAGAGGCTTCTCTGAGAAAGAAAGATGTCGAATGGGTATTCCACAGACGAGAATTTCCGCTATCTC 6001---------+---------+---------+---------+---------+---------+ 6060GAAGAGACTCTTTCTTTCTACAGCTTACCCATAAGGTGTCTGCTCTTAAAGGCGATAGAGATCTCGTGCTTCAGGGCCAGGGTGAAAATGTACATCCAGGTGGAGCCTGTGCTGGACTAC 6061---------+---------+---------+---------+---------+---------+ 6120TAGAGCACGAAGTCCCGGTCCCACTTTTACATGTAGGTCCACCTCGGACACGACCTGATGCTGACCTTTCTGCCTGCAGAGGTGAAGGAGCAGATTCAGAGGACAGTCGCCACCTCCGGG 6121---------+---------+---------+---------+---------+---------+ 6180GACTGGAAAGACGGACGTCTCCACTTCCTCGTCTAAGTCTCCTGTCAGCGGTGGAGGCCCAACATGCAGGCAGTTGAACTGCTGCTGAGCACCTTGGAGAAGGGAGTCTGGCACCTTGGT 6181---------+---------+---------+---------+---------+---------+ 6240TTGTACGTCCGTCAACTTGACGACGACTCGTGGAACCTCTTCCCTCAGACCGTGGAACCA      EcoRI         | TGGACTCGGGAATTCGTGGAGGCCCTCCGGAGAACCGGCAGCCCTCTGGCCGCCCGCTAC6241 ---------+---------+---------+---------+---------+---------+ 6300ACCTGAGCCCTTAAGCACCTCCGGGAGGCCTCTTGGCCGTCGGGAGACCGGCGGGCGATGATGAACCCTGAGCTCACGGACTTGCCCTCTCCATCGTTTGAGAACGCTCATGATGAATAT 6301---------+---------+---------+---------+---------+---------+ 6360TACTTGGGACTCGAGTGCCTGAACGGGAGAGGTAGCAAACTCTTGCGAGTACTACTTATA                                 HindIII                            BstXI      |                                |      |CTCCAACTGCTGAACCTCCTTCAGCCCACTCTGGTGGACAAGCTTC 6361---------+---------+---------+---------+------ 6406GAGGTTGACGACTTGGAGGAAGTCGGGTGAGACCACCTGTTCGAAG

REFERENCES

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1-35. (canceled)
 36. A melanoma differentiation associated gene-5protein having SEQ ID NO:2.
 37. A cell containing a nucleic acidsequence comprising a MDA-5 promoter linked to a heterologous reportergene.
 38. A method for identifying compounds that effect regulation ofMDA-5 expression, comprising testing for the ability of a compound toactivate reporter gene expression in a reporter cell containing anucleic acid sequence comprising a MDA-5 promoter linked to aheterologous reporter gene, wherein an increased level of expression ofreporter gene indicates that the compound is an interferon agonist.